Probiotics may be an ideal choice for these patients, given it can improve the defecation and quality of life of individuals with chronic diarrhoea. However, evidence-based medical research is still limited to support its use as a diarrhoea agent.Lactobacillus plantarum p9 probiotics powder) or a placebo group. Except an independent project administrator who will be responsible for unblinding, the other researchers are blinded. Primary outcome is diarrhoea severity score, and secondary outcomes include weekly mean frequency of defecation, weekly mean stool appearance score, weekly mean stool urgency score, emotional state score, gut microbiome, and faecal metabolome. Each outcome measure will be assessed at the timepoints of pre-administration (day 0), administration (day 14 and/or 28), and post-administration (day 42) to identity inter- and intra-groups differences. Adverse events will be recorded to evaluate the safety of L. plantarum p9.A randomized, double-blind, placebo-controlled clinical trial is designed to pinpoint the efficiency and possible action modes of probiotics for chronic diarrhoea. 200 eligible volunteers with chronic diarrhoea are randomly assigned to a probiotic group  (NO. ChiCTR2000038410). Registered on November 22, 2020,  And an Probiotics always hold the advantages of safety and widely public acceptance . Evidenc22.1L. plantarum p9 or placebo at home and are followed up online via completing designed online questionnaires. For the stool sample collection, volunteers are required to immediately inform researchers as soon as prepared according to instructions, and the researchers timely take it back for further detection. This paper is written according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) ) 2.3(1)Personal or family history of colon cancer, celiac disease, or inflammatory bowel disease.(2)Confirmed Intestinal organic diseases by colonoscopy.(3)Plans to be pregnant with or father a child in near future, or at the status of pregnancy or breastfeeding in women.(4)plantarum p9 or placebo.Allergies to L. (5)Intaking antibiotics or probiotics within the past two weeks.(6)Intaking antianxiety, antidepressant, or other psychotropic drugs within the past month.(7)Need for long-term use of medications for diarrhoea.(8)History of severe diseases, such as myocardial infarction, cerebral infarction, and malignant tumour, judged by the investigators as disqualifying conditions.(9)Major mental illnesses, inability to control one's actions, or inability to cooperate.(10)Illiteracy, inability to understand the informed consent form, or inability to independently sign the informed consent form.Volunteers have one of the following situations are excluded.2.4The investigators recruit volunteers from the public via oral communication, posters, and dissemination of WeChat official account. With the help of WeChat, a two-dimensional (QR) code is available for volunteers to scan and register for enrolment, submitting demographic data, and answering questions related to inclusion and exclusion criteria. Then, the potentially eligible volunteers will undergo three rounds of screening, as follows.First, the registration information is collected and collated, according to which potentially eligible candidates are invited to participate in a face-to-face consultation with clinicians and researchers for the further screening.Second, a series of face-to-face consultations are organized: 1) Trained researchers introduce the trial to the potentially eligible volunteers through video and information sheets regarding the main aspects of the trial, especially the content of informed consent materials, and discuss the trial with the potentially eligible volunteers. 2) Then, the clinicians also have an informed face-to-face consultation with the potentially eligible volunteers to confirm the registered information, further selecting out potentially eligible volunteers according to inclusion and exclusion criteria to proceed in the next procedure. The selected potentially eligible volunteers are essential to sign an informed consent form directly with a member of data management team (DMT) before proceeding into the next procedure.Third, the potentially eligible volunteers undergo a 14-day screening period. During the third round of selecting, the potentially eligible volunteers are not allowed to take any medicines or health products to improve their diarrhoea symptoms. And each volunteer will be asked to collect one stool sample and complete an online defecation diary . At the 2.5Based on the order of registering during above recruitment, eligible volunteers are given a serially unique number, i.e., 001, 002, 003, 004, 005, and so on. The unique number is used as volunteer's identification card throughout the whole research periods instead of volunteer name or any other information, so as to guarantee confidentiality for volunteers. For each unique number, a random sequence is automatically generated using the computer software by two independent project administrators. The unique number with an odd random sequence is assigned into probiotic group, while the unique number with an even random sequence is assigned into placebo group. During the study, except two independent project administrators, the other participants of the study, including volunteers, treatment packs distributors, data collectors and analysts are blinded to the randomization sequence, namely the grouping. The randomization sequence is maintained by two independent project administrators and will only be unblinded in the case of major safety issues or when performing the interim or final data analysis. Moreover, the probiotics or placebo treatment packs are labelled with the foregoing unique numbers according to the grouping of the unique numbers, and the treatment packs distributors distribute the treatment packs according to the unique number on the packs to the corresponding volunteers, in such a way, allocation concealment is achieved.2.6L. plantarum P9. L. plantarum P9 powder comprises L. plantarum P9 (40%), maltodextrin (20%), orange powder (20%), and maltitol (20%). The placebo comprises maltodextrin (60%), orange powder (20%), and maltitol (20%) and has the same appearance, packaging, and taste as the L. plantarum P9 powder. Both probiotics and placebo packs are stored in a 4 °C, dry place away from direct sunlight. At the end of the phase, all unused probiotics or placebo and used (empty) packs are collected, so as to calculate volunteers’ compliance.One probiotics or placebo powder pack per day. One probiotics powder pack contains 100 billion colony-forming units (CFU) of (1)Period of screening (observation period of pre-administrating) (days −14 to 0): volunteers do not receive probiotics or placebo. Eligibility screening are performed as described in “volunteer recruitment part” .(2)L. plantarum P9 powder with warm boiled water (below 40 °C) on a full stomach, 1 pack per day ; if volunteers have to take antibiotics during the administration, the probiotics is taken 2 h later. 2) For placebo group: volunteers take the placebo in the same manner as probiotics group.Observation period of administrating (days 0–28): 1) For probiotics group: volunteers take (3)Observation period of post-administrating (days 29–42): No interventions are taken during the study.The study is designed to include three phases, a period of screening (an observation period of pre-administrating), an observation period of administrating, and an observation period of post-administrating . The int2.7The following interventions or factors may confound the results of the study and are prohibited: 1) other kinds of probiotics, prebiotics, and foods containing probiotics (such as yoghurt); 2) antianxiety, antidepressant, and other psychotropic drugs; 3) other substances may benefit intestinal symptoms. Furthermore, antibiotics are not advised, and normal dietary habits are recommended during the study. All concomitantly used substances/drugs related to defecation are required to record with an explanation via online daily diary .2.8The primary outcome is the diarrhoea severity score, which is set as to the gastrointestinal symptom rating scale (GSRS) . The outA defecation diary developed with reference to the literatures ,34,37 wi2.9(1)Similarly, based on the diary , the wee(2)The Depression, Anxiety and Stress Questionnaire (DASS-21)  Appendi will be (3)For the indicator of gut microbiome, DNA are extracted from stool sample using the QIAamp Fast DNA Stool Mini Kit . An agarose gel electrophoresis and Nanodrop spectrophotometer are used to examine the quality of DNA. Then, shotgun metagenomic sequencing are performed for all samples using an Illumina HiSeq 2500 instrument. And sequence libraries are constructed by DNA fragments of ∼300 bp length which obtained from paired-end reads of ∼150 bp lengths generated by sequencing in bi-directions. In addition, the metagenomic analysis is performed from the following aspects: analysing alpha diversity and beta diversity in each group to determine the intra-groups differences in the microbiota compositions; analysing the inter- and intra-groups taxonomic characteristics at the levels of phylum, genus, and species to identify specific genes related to diarrhoea. Metagenomic biological pathway analysis are used to evaluate the effect of probiotics on the function of the gut metagenomics in patients with diarrhoea and hopefully to explore the metagenomic biological pathways contributing to the mechanism of probiotics on the treatment of diarrhoea.(4)For the indicator of faecal metabolome, briefly, stool samples treated by protein precipitation method and the supernatant are used for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The original data is subjected to peak alignment, retention time correction and peak area extraction through the XCMS-Plus program. The structure of metabolites is identified by accurate mass matching (<5 ppm) and two-level spectrum matching, and METLIN database is retrieved. After deleting the missing value > 50% and normalizing the data, multi-dimensional statistical analysis is performed, like unsupervised principal component analysis (PCA), supervised partial least squares discriminant analysis (PLS-DA) and potential difference metabolite analysis. Metabonomic analysis can further identify the potential differential metabolites of probiotics in treatment of diarrhoea and examine the correlation analysis between the gut microbiota and metabolites.The secondary outcome measures include weekly mean frequency of defecation, weekly mean stool appearance score, weekly mean stool urgency score, emotional state score, gut microbiome, and faecal metabolome.2.10L. plantarum P9 powder will be evaluated as the following four levels: excellent (safe with no AE); good (relatively safe with moderate AEs that disappear on their own without any specific treatment); conditional ; and unsafe  , where k is the number of sample groups in the comparison. More detailed information about how to analyse and present inter-group differences is clearly provided in Then, to identify the intra-group changing trends with the consecutive phases, namely, pre-, during and post-probiotic administrating, the P-value will be generated based on 999 permutations. t-test, Wilcoxon test and Kruskal-Wallis test will be used to evaluate differences in various variables between and within groups; P values will be corrected for multiple testing using the Benjamini-Hochberg procedure. Meanwhile, Pearson and spearman correlations will be used to analyse the correlations between different indicators in clinical, metagenomics and metabolomics.In addition, the statistical analyses of metagenomics and metabolomics will be performed using the R software (v.4.0.2) and Adobe Illustrator. PCA and PLS-DA analysis will be performed and visualized using the R package vegan, ggplot and ggpubr, and the adonis 3Chronic diarrhoea can disturb the patient's quality of life, work performance and wellbeing, increase patient's expenses , and leaL. plantarum p9, belongs to Lactobacillus species, which is one of the most studied probiotics [Clostridium difficile-associated diarrhoea [Some special steps are taken to guarantee the eligibility of volunteers, the feasibility of study, and interpretability of results, including completing diary online daily , face-to-face consultation with the clinical specialist , and stool test  in the 14 days screening period. The study intervention, obiotics ,42 and riarrhoea ,43 and tiarrhoea .L. plantarum P9 on defecation and quality of life in individuals with chronic diarrhoea. Moreover, the study will try to explore the possible action modes of probiotics to improve chronic diarrhoea, from the aspects of gut microbiome and faecal metabolome. Gut microbiome has been found to be altered in chronic diarrhoea patients and may be a cause of chronic diarrhoea [L. plantarum P9 on chronic diarrhoea. Thus, the set of multidimensional outcomes will form a chain of evidence.Multidimensional outcomes are examined to produce a chain of evidence from causal improvements in metagenomic and metabolomic data to clinical symptoms and then to the emotional state. Chronic diarrhoea can disturb the patient's quality of life, work performance and wellbeing ; therebyiarrhoea . Probiotiarrhoea ,26. The A limitation is the way of follow up in this study (completing diary online daily), which may lead to bias about the effectiveness of probiotics. To overcome the limitation, a WeChat group is established for the cohort of volunteers, so the researcher can timely post messages via the WeChat group, keeping a good compliance; and an DMT is formed to promptly assess the study quality and safety. during the assessment, once an uncertainty about the data arises, the DMT can urge the data collectors to resolve it. The confounding factors are also prohibited. In such a way, the bias would be minimized as far as possible.L. plantarum p9 can improve the defecation and well-being of individuals with chronic diarrhoea. If the study findings are as expected, L. plantarum p9, taken as probiotics, may become an optimal nonmedical intervention option for patients with chronic diarrhoea.Collectively, the study protocol will provide high-quality evidence for the use of probiotics as a diarrhoea agent when it is strictly conducted out, providing evidence regarding whether and to what extent 4This study started recruiting patients on October 1, 2020. To date, 174 patients have been enrolled. The enrolment is expected to be completed by October 31, 2022, and all follow-ups are expected to be completed by December 31, 2022. The protocol is 2.0 version, and the date of edition was September 10, 2020.Not applicable.This study was registered at Chinese Clinical Trial Registry (ChiCTR) (NO. ChiCTR2000038410) on November 22, 2020 , and appData sharing is not applicable to this trial as no database is generated or analysed for the current study. And when the study completed, the study results will be released to the public, volunteers, and the general medical community via publishing journal article, with all the related data being available. The process of publication will be independent from the funder and sponsor.National Natural Science Foundation of China (31720103911) to Heping Zhang . Jiangzhong Pharmaceutical Company Limited also is a sponsor and can be contacted to Jianhua Wan at email: wanjianhua@crjz.com. The funders play no role in the design of study, data collection and analysis, or preparation of the manuscript.The trail is supported by the ng Zhang . L. planWJL, The authors declare that they have no competing interests. And the study has not received funding/assistance from a commercial organization.


Venous thromboembolism (VTE) is the second leading cause for death of radical prostatectomy. We aimed to establish new nomogram to predict the VTE risk after robot-assisted radical prostatectomy (RARP).Patients receiving RARP in our center from November 2015 to June 2021, were enrolled in study. They were randomly divided into training and testing cohorts by 8:2. Univariate and multivariate logistic regression (model A) and stepwise logistic regression (model B) were used to fit two models. The net reclassification improvement (NRI), integrated discrimination improvement (IDI), and receiver operating characteristic (ROC) curve were used to compare predictive abilities of two new models with widely used Caprini risk assessment (CRA) model. Then, two nomograms were constructed and received internal validation.p < 0.05). The C-index of Model A and Model B were 0.968 and 0.978, respectively. For clinical usefulness, the two new models offered a net benefit with threshold probability between 0.08 and 1 in decision curve analysis, suggesting the two new models predict VTE events more accurately.Totally, 351 patients were included. The area under ROC of model A and model B were 0.967  and 0.978 , which also were assayed in the testing cohorts. Both the prediction and classification abilities of the two new models were superior to CRA model (NRI > 0, IDI > 0, Both two new models have good prediction accuracy and are superior to CRA model. Model A has an advantage of less variable. This easy-to-use model enables rapid clinical decision-making and early intervention in high-risk groups, which ultimately benefit patients.The online version contains supplementary material available at 10.1245/s10434-022-11574-5. Actually, the risk of postoperative thrombosis events varies with different tumors.15 However, there are still limited studies on using nomograms to predict the risk of thrombosis events.16 In this study, we established two nomograms to predict the risk of VTE after robot-assisted radical prostatectomy (RARP). Referring to these models, we believed that doctors could identify patients with a high risk of VTE and take timely preventive measures and treatments at an early stage, which enable to help reduce the risk of postoperative thrombotic events after RARP.Nomogram is a visible way to show the regression results, which can express the significance of different variables on the outcome by the length of the line. By calculating the total score, the probability of the outcome in the patient can be visually displayed.A total of 351 patients receiving RARP in the Xiangya Second Affiliated Hospital, Central South University from November 1, 2015, to June 1, 2021, were enrolled in this study. All of them are Han Chinese, and no separate emphasis in the Table In this study, all patients were diagnosed with prostate cancer according to the histopathologic examination and the diagnosis of VTE was confirmed by a positive finding on color duplex ultrasound for DVT or computerized tomography pulmonary angiography (CTPA) for PE. All ultrasound/CTPA images were explained by two senior radiologists. We undertook a radical screening strategy to identify VTE. D‐Dimer test was given to all patients after RARP, lower limb ultrasound was given when patients with positive D‐Dimer test, and ultrasound/CTPA was given in patients with VTE-related symptoms. These examinations were performed with the permission of patients.17 Finally, patients were allocated into the training cohort (n = 246) and the testing cohort (n = 105), respectively. In addition, VTE event was an endpoint of the one-month follow-up after surgery, and the above patients were finally divided into the VTE and non-VTE groups.All patients were randomly distributed to the training and testing cohort with 7:3 by using computer-generated random numbers.19 We collected the basic information , laboratory data , imaging results (prostate volume), and treatment measures  from the hospital medical records. Of note, patients with intermediate and high-risk of biochemical recurrence were selectively undergone pelvic lymphadenectomy. If a lymph node dissection was deemed necessary, an extended lymph node dissection was mostly performed. Besides, all patients were scored with Caprini risk assessment on admission to assess the risk of VTE during the perioperative period.20 Patients with a caprine score of 3–4 was given mechanical prophylaxis until ambulation, while those with a caprine score more than 5 was given pharmacologic prophylaxis after surgery, according to the recommendationsCandidate clinical variables were selected based on published studies, accessibility, and professional knowledge.We first performed univariate logistic regression analysis on candidate variables and then performed multivariate logistic regression analysis on variables with significant differences to select independent risk factors. A nomogram model was accordingly constructed based on the independent risk factors. Meanwhile, to get the optimal model, we also established another evaluation model by package “MASS” and stepwise logistic regression was used to obtain clinical variables for model construction.21:Receiver operating characteristic curve (ROC) was generated, and the area under the curves (AUC) was used to measure discrimination. Meanwhile, the Brier score, which was calculated as the following formula, was employed to test calibrationN forecasted instances; ft forecasted probability; ot true event.After constructing the two new models by using the univariate and multivariate logistic regression analysis (model A) and stepwise logistic regression analysis (model B), we calculated the calibration and discrimination in the primary training dataset.22 Finally, for ease to use clinically, nomograms were constructed for each new model fitted in the above regression methods.Then AUC, Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) were used as indicators to compare predictive abilities of two new models and Caprini risk assessment (CRA) model. “pROC,” “nricens,” and “PredictABEL” packages were utilized to process the analysis above. The method of Bootstrap was used to self-validate by 1000 repetitions and followed with another internal validation in the testing cohort. The C-index was calculated by use of the RMS package.t-test, and the count data were compared by chi-square test or Fisher’s exact test. Two-sided p < 0.05 was considered as statistical significance. All statistical analyses were conducted through SPSS (version 26.0) and R software (Version: 4.1.0).In this study, variables with more than 30% missing values were discarded, and the missing data were filled by predictive mean matching algorithm. The measurement data were presented in the form of mean with standard deviation or median with interquartile range, and the count data were presented in the form of a number (percentage). The measurement data were compared by two unpaired sample Table p < 0.001), a larger prostate volume  with a higher PSA level , and a higher Gleason score , which usually indicated a poor prognosis. In addition, the VTE group experienced longer operation time  and more lymph node dissection . It is interesting to note that, in our study, many patients in the VTE group had a clear previous history of VTE , and had a higher coagulation index  before surgery  and coagulation index (p < 0.001) were screened as independent predictors for VTE after RARP  to select the significant predictors and a combination containing 12 variables is initially enrolled. As a result, age, coagulation index, prostate volume, neoadjuvant ADT, tumor stage, Gleason score, PSA level, preoperative APTT, and postoperative D-Dimer were finally selected for model construction. For all of the above, The ROC was conducted to assess the discrimination performance of established models. Model B was shown to be the best with an AUC of 0.988 , whereas model A was not inferior, with an AUC of 0.957 (95% CI 0.928–0.985). The AUC of Model A and Model B was both obviously higher than the CRA model . Those results also were confirmed in the testing cohort with AUCs of 0.869 (95% CI 0.769–0.969), 0.863 (95% CI 0.753–0.973), and 0.777 (95% CI 0.644–0.910), respectively  with significant statistical differences  was conducted to evaluate the clinical usefulness of the two prediction nomograms. The two new models offered a net benefit at the threshold probability between 0.08 and 1 in the training cohort Fig. A. Althou25 but prostate cancer patients with VTE tend to indicate a poor prognosis and shorter life expectancy.12 In addition, they also have an increased risk of recurrent venous thromboembolism and severe bleeding complications.12 Therefore, it is important to identify high-risk patients and take early intervention to reduce the incidence of VTE after RARP. Nevertheless, it is not easy to predict the risk of VTE. The tumor was heterogeneous itself, and the risk of VTE was related to the interaction among tumor cells, coagulation system, and clinical characteristics of patients.12 A precise predicting tool special for each type of cancer may greatly do a favor to clinicians to predict postoperative VTE. However, at present, most of the references are based on the investigation for risk factors of postradical prostatectomy, still lacking accurate prediction models for individual VTE risks.26The risk of VTE after RARP is not very high,11 Among them, Gleason score is a widely used method for histological grading of prostate cancer in current clinical guidelines. Usually, the higher the score, the worse the prognosis.27 In addition, prostate volume is associated with the grading and prognosis of prostate cancer.28 These results indicate that the increase of VTE is an inherent biological characteristic of some tumors. Research by Cronin-Fenton et al. confirmed that in their study cohort, the VTE risks for the special cancers varied even after adjusting for all the possible confounders.29 The risky factor includes hypercoagulable state caused by tumor, vascular damage caused by treatment, surgery, and tumor itself.31 Noteworthy, cancer patients tend to receive more medical intervention and a forced sedentary lifestyle, both of which increase the risk of venous thromboembolism. Larger prostate volume, neoadjuvant ADT, and advanced tumor stage mainly cause the longer operation time, which also is an important factor affecting the risk of VTE after RARP, in accordance with the findings of Abel et al.10 Published researchers have elaborated that excessive operation time would increase stress response, change coagulation status, and increase the risk of thrombosis. In addition, anesthesia time would increase due to prolonged operation time. Thus, the anesthesia would slow down the patient's blood flow, and the anesthetic drug may damage the patient's vascular endothelial cells as well, leading to the occurrence of thrombosis.33 Tasaka et al. have proved that patients older than aged 60 years have an increased risk of postoperative thrombosis.34 Elderly patients usually have higher blood viscosity, thicker intima, impaired cardiopulmonary circulation, and decreased immunity (susceptible to infection), making them more likely to develop thrombosis.35 In addition, elderly patients recover more slowly and stay in bed for a longer time, which also greatly increases the risk of thrombosis. It is worth noting that in our study, only 1.3% (4/311) of patients in the non-VTE group had a previous history of thrombosis, compared with 35.0% (14/40) in the VTE group, which showed significant statistical difference in univariate analysis (p < 0.001), and suggested that there is a strong correlation between the risk of postoperative VTE and previous history of thrombosis in patients with prostate cancer. Although it may be a strong independent predictor, this factor was not finally included in the model (p > 0.05). The reason could be that the two new models constructed by other combined variables would be closer to the actual prediction results. In this study, the median PSA level and ranges are very high when compared with literatures, one reason could be the higher percentage of advanced prostate cancer in China and second could be the neoadjuvant ADT used here. In addition, 11.4% patients developed VTE in this study, which is surprisingly higher than that in previous reports.6 However, it is plausible considering the greater attention given to postoperative venous thromboembolism prophylaxis in our center and more patients received VTE screening as mentioned above.Our study shows that the risk of post-RARP VTE in patients is mainly related to the following factors: age, coagulation index, prostate volume, neoadjuvant ADT, tumor stage, Gleason score, PSA level, preoperative APTT, postoperative D-Dimer, and Gleason score, which is mostly consistent with previous reports of risk factors for postoperative thrombosis.20 which has been demonstrated in patients undergoing different surgeries, of which urological surgery accounts for 17% of the study population.36 There are more than 30 indicators used to assess the risk of thrombosis of patients; however, it seems too complicated to use, not conducive to clinical fast decisions. In addition, due to the uniqueness of prostate cancer, Caprini score’s universal standards may not be very appropriate for the risk assessment of prostate cancer. Furthermore, the models obtained by stepwise regression and U&M regression in this study have respectively eight and three variables, which greatly simplifies the risk assessment process of VTE. Model A has an advantage of less variable. The AUC of the two new models is significantly higher than the CRA model, besides, both NRI and IDI > 0, which shows better predictive capabilities. The advantages of the visualization of the nomogram also improve the convenience of clinical applications and do a favor to clinical evaluation and management, early thromboprophylaxis would be suggested when a risk of VTE was calculated by the nomogram. Notably, the applied statistical methodology could be overfitted due to the limited study size.Caprini score is a common way to evaluate patients at risk of VTE,Indeed, our study still has some limitations of the following aspects. First, the prediction models in one clinic does not necessarily have to be useful in another. One way forward is to look for causal associations and to let each center make its own prediction models with the support of evidence for causality. Second, this is a retrospective, observational, single-center, retrospective study with limited study size, which is easy to produce sampling error and needs to be verified by the data of larger samples. Lastly, by use of a nomogram, which kind of thromboprophylaxis should be taken to maximize the benefits still needs further study.By the retrospective analysis of prostate cancer data from our center, two sets of prediction models were developed. Both two new models have good prediction accuracy and are superior to the well-known CRA model in our clinic. Model A has an advantage of less variable. This easy-to-use nomogram may help to rapid clinical decision-making for urologists and early thromboprophylaxis in high-risk groups that ultimately benefit patients, which encourage the researcher to make a more stable and universal prediction nomogram of VTE for RARP.Supplementary file1 (TIF 160 kb)Supplementary file2 (TIF 2262 kb)Supplementary file3 (DOCX 33 kb)Below is the link to the electronic supplementary material.


Immune escape and metabolic reprogramming are becoming important characteristics of tumor biology, which play critical roles in tumor initiation and progression. However, the integrative analysis of immune and metabolic characteristics for the tumor microenvironment in hepatocellular carcinoma (HCC) remains unclear. Herein, by univariate and least absolute shrinkage and selection operator (LASSO) Cox regression analyses, a prognostic signature associated with tumor microenvironment was established based on five immune- and metabolism-related genes (IMRGs), which was fully verified and evaluated in both internal and external cohorts. The C-index was superior to previously published HCC signatures, indicating the robustness and reliability of IMRGs prognostic signature. A nomogram was built based on IMRGs prognostic signature and various clinical parameters, such as age and T stage. The AUCs of nomogram at 1-, 3-, and 5-year  were slightly better than that of IMRGs signature . The relationship of risk score (RS) with immune checkpoint expressions, immunophenoscore (IPS), as well as microsatellite instability (MSI) together accurately predicted the treatment efficacy. Collectively, the IMRGs signature might have the potential to better predict prognostic risk, evaluate immunotherapy efficacy, and help personalize immunotherapy for HCC patients. Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors globally with a high morbidity and mortality rate , and theImmune escape  and metaIn this study, a systematic and comprehensive integrative analysis of immune- and metabolism-related genessignature was constructed in HCC, and the prognostic value was analyzed. Moreover, a prognostic nomogram was developed to provide a quantitative analysis tool in order to predict prognostic risk in HCC patients.https://portal.gdc.cancer.gov/) and ICGC database (https://dcc.icgc.org/). According to the ratio of 7:3, the TCGA-LIHC participants were randomly assigned to two cohorts: the training cohort (N = 262) and the testing cohort (N = 108). The clinical characteristics of the two cohorts were summarized in https://www.immport.org). The metabolism-related genes were extracted by downloading the “c2.cp.kegg.v7.4.symbols” from MSigDB (Version 7.4).Clinical features and gene expression profilesof HCC samples were downloaded from TCGA  Cox regression analysis. Accordingto the median risk score (RS), the TCGA training cohort was categorized into high- and low-risk groups. The ICGC-LIRI-JP cohort was subsequently analyzed in line with the cutoff value on the TCGA training set.via “rms” R package.On the basis of IMRGs prognostic signature and various clinical parameters, a prognostic nomogram was constructed to predict the survival probability of HCC patients. The predictive performance of the nomogram was evaluated by comparing predicted and actual survival risks. The calibration curves at 1-, 3-, and 5-year were plotted https://tcia.at/home), which was used to predict the therapeutic responses to the four major immune checkpoints . Moreover, we analyzed the correlation of IMRGs signature with microsatellite instability (MSI), an indicator used to reflect the efficacy of immunotherapy.Immunophenoscore (IPS) was calculated using the four main factors, including MHC molecules, immunomodulators, effector cells, and suppressor cells, in theTCIA database   curve and the area under the curve (AUC).R software (version 4.0.3) was applied to conduct the statistical analyses with + T cells, NK cells as well as a high proportion of monocytes-macrophages and neutrophils.After filtering and deduplication, a total of 2,715 immune- and metabolism-associated genes (IMRGs) were included. Compared with the normal group, 546 DEGs from IMRGs were observed in HCC samples by further difference analysis, with 441 DEGs upregulation and 105 DEGs downregulation  and external validations (ICGC-LIRI-JP cohort) were performed. The RS of validation groups was determined according to the same formula as the TCGA training set. Likewise, the high- and low-risk groups were classified using the same cutoff value as the training group.P = 0.002), entire TCGA-LIHC (P < 0.001), and ICGC cohorts  for the high-risk  = 1.201 (1.135–1.272), P value less than 0.1 were determined by multiple regression, namely, age, T stage, and RS, and a nomogram was constructed to predict survival risk for individuals , a member of the class I cytokine receptor superfamily, was down-regulation in the high-risk group and was related to chemoresistance, tumor metastasis, and poor prognosis –29. Acylt manner  and coult manner , 35. POLt manner . HoweverThe advent of immunotherapy has provided new ideas for the treatment of HCC, of which immune checkpoint inhibitors (ICIs) have become a potentially effective therapeutic strategy –38. The There are several strengths in this research as follows: First, the robustness and reliability of IMRGs prognostic signature were evaluated and validated using multiple datasets, including internal and external cohorts. Second, the associations of RS with immune checkpoint expressions, four IPS scores, as well as MSI were comprehensively and deeply explored. Third, a nomogram for quantitative calculation was developed in order to assist with clinical application. Nevertheless, there are still several limitations in this study. For example, The IMRGs prognostic signature and the nomogram were established based on a retrospective study, which needs to be further verified in large multicenter prospective cohorts.The IMRGs prognostic signature was constructed based on the integrated analysis of immune- and metabolism-related genes, which could better predict prognostic risk and the response to immunotherapy. We also developed a nomogram for patients with HCC, providing an effective quantitative analysis tool to realize the clinical application of personalized precision therapy.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/XT, XZ, and QK conceived and directed the completion of the study. HG collected and downloaded the data. YG conducted the data analysis and drafted the manuscript. JY modified the language and revised the manuscript. All authors contributed to the manuscript and approved the submitted version.This study was funded by the Medical Science and Technology Research Plan Joint Construction Project of Henan Province, China (No. LHGJ20210282) and the National Key Research and Development Program of China (No. 2020YFC2008304).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.


Meanwhile, camel milk had a positive modulatory effect on the intestinal flora of NAFLD mice, increasing the relative abundance of beneficial bacteria and decreasing the relative abundance of harmful bacteria in the intestinal flora of NAFLD mice, and silymarin had a similar modulatory effect. At the genus level, camel milk increased the relative abundance of Bacteroides, norank_f_Muribaculaceae and Alloprevotella and decreased the relative abundance of Dubosiella and Coriobacteriaceae_UCG-002 (p < 0.05). Camel milk also enhanced Carbohydrate metabolism, Amino acid metabolism, Energy metabolism, Metabolism of cofactors and vitamins and Lipid metabolism in NAFLD mice, thus reducing the degree of hepatic lipid accumulation in NAFLD mice and maintaining the normal structure of the liver. In conclusion, camel milk can improve the structure and diversity of intestinal flora and enhance the levels of substance and energy metabolism in NAFLD mice, which has a positive effect on alleviating NAFLD and improving the structure of intestinal flora.Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disease of life, usually caused by unhealthy diet and lifestyle. Compared to normal individuals, the structure of the intestinal flora of NAFLD patients is altered accordingly. This study investigates the effect of camel milk on the regulation of intestinal flora structure in mice with high-fat diet-induced NAFLD. NAFLD model was established by feeding C57BL/6J mice a high-fat diet for 12 weeks, meanwhile camel milk (3.0 g/kg/d), cow milk (3.0 g/kg/d), and silymarin (200 mg/kg/d) were administered by gavage, respectively. Food intake and changes of physiological indexes in mice were observed and recorded. The 16S rRNA gene V3-V4 region was sequenced and the intestinal flora diversity and gene function were predicted in the colon contents of mice from different group. The results showed that camel milk enhanced glucolipid metabolism by downregulate the levels of blood glucose and triglyceride (TG) in serum, reduced lipid accumulation by downregulate the level of TG in the liver and improved liver tissue structure in NAFLD mice ( Non-alcoholic fatty liver disease (NAFLD), a major community health challenge for the World Health Organization, is one of the most common and preventable liver diseases, affecting approximately one-quarter of the population, and is expected to become a worldwide epidemic in the future , 2. AccoCamel milk, known as “desert platinum” because of its minimal production, is a nutritionally balanced food with various biological functions such as anti-allergy, antioxidant, and antihypertensive . BecauseAllobaculum and Akkermansia and a decreased Erysipelotrichaceae and Desulfovibrionaceae  are important factors affecting the health of the body, and changes in the structure of the intestinal flora often mean the onset of disease , 27. In ionaceae . TherefoIn this study, NAFLD model was established by feeding C57BL/6J mice with high-fat diet for 12 weeks. Meanwhile, camel milk, cow milk, and silymarin were gavaged daily to mice in camel milk group, cow milk group and silymarin group, respectively. The changes of the physiological and biochemical indexes of NAFLD mice were observed to study the effects of camel milk, cow milk and silymarin on the alleviation of NAFLD and the intestinal flora of mice.Total triglycerides (TG) kit (Item No. A110-1-1) and Total cholesterol (TC) kit (Item No. A111-1-1) were purchased from Nanjing Jiancheng Institute of Biological Engineering. ELISA kit obtained from Shanghai EnzymeLink Biotechnology Co. Silymarin and carboxymethyl cellulose were purchased from Shanghai Yuanye Biological Co.–1, 30 min) to separate the fat, freeze-dried, sieved and packaged, and stored at 4°C. High-fat feed  was purchased from Nantong TROPHY Feed Technology Co. Camel milk, cow milk and silymarin were mixed with 0.5% carboxymethylcellulose (CMC), respectively.Camel milk and cow milk were collected from the pasture area of Alax Right Banner, Alax League, Inner Mongolia, China. Fresh camel milk was transported back to the laboratory after quick-freezing in liquid nitrogen, and stored at −80°C. Frozen camel milk was thawed in cold water, centrifuged  Biotechnology Co., Ltd. Animals were housed in IVC animal experimental system with a housing temperature of 20°C ± 1°C, relative humidity of 50% ± 5%, and a 12-h day/night cycle. Mice were acclimatized and fed for 7 days prior to the start of the test, with free access to food and sterile distilled water. The control diet was gradually replaced with high-fat diet during acclimatization feeding. All animal experiments are conducted in strict accordance with the guidelines for the care and use of laboratory animals as described by the National Institutes of Health, minimizing the amount of mice and reducing their suffering while achieving the experimental objectives. The experimental program was approved by the Ministry of Education Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University (License No.: SYXK Meng 2020-0002). During the experimental period, the NC group was fed the control diet, and the other groups were fed high-fat feed.n = 10), model group , camel milk group , cow milk group , and positive control group . After 12 weeks, the NAFLD model that was characterized by increased liver index and the levels of TG and TC in the liver was established. To alleviate NAFLD in mice, camel milk, cow milk and silymarin were administered by gavage. Twice daily (9:00 a.m and 4:00 p.m), CaM group was gavaged with camel milk (3.0 g/kg/d), CoM group was gavaged with cow milk (3.0 g/kg/d), PC group was gavaged with silymarin (200 mg/kg/d), Mod group and NC group were gavaged with 0.5% CMC (0.2 ml/d/per)  . The oraBody weight, blood glucose, liver index, and the levels of TG and TC in the liver are important indices. Body weight and blood glucose of mice were measured weekly, blood glucose was detected using a blood glucose meter. Feed consumption was weighed and recorded every 3 days. The liver was weighed after dissecting the mice and the liver index was calculated. Liver was used to determine TG level and TC level and to make pathological sections (hematoxylin eosin staining) for observing the pathological structure of the liver. Liver tissue samples stored in 4% paraformaldehyde were gradient dehydrated and paraffin-embedded, and then cut into 5-μm sections. The sections were stained with hematoxylin staining solution and eosin staining solution, dried and placed under a microscope to observe the liver tissue structure. Liver index is calculated as follows.The levels of alanine transaminase (ALT), aspartate aminotransferase (AST), leptin (LEP), adiponectin (ADPN), high density lipoprotein cholesterol (HDL-c), low density lipoprotein cholesterol (LDL-c), interleukin (IL-6), tumor necrosis factor-alpha , and insulin (INS) in serum were measured according to manufacturer’s instructions by ELISA kits . And the levels of total triglycerides (TG) and total cholesterol (TC) in serum and liver were detected, respectively.After 12 weeks of gavage, mice were fasted without water for 12 h and gavaged with 30% glucose solution (1.5 g/kg), then the blood glucose values were measured at 0, 30, 60, 90, and 120 min. Mice were given intraperitoneal injection of insulin (0.5 U/kg), and the blood glucose values at 0, 30, 60, 90, and 120 min were detected. While the area under the curve of ITT and OGTT were calculated. According to the formulas, the insulin resistance index (HOMA-IRI) and insulin sensitivity index (HOMA-ISI) were calculated .Fins is mice fasting insulin, FBG is mice fasting blood glucose.To observe the differences in intestinal flora different groups of mice, fresh fecal was collected in lyophilization tubes and quick-frozen in liquid nitrogen during week 12, and DNA of mouse intestinal flora was extracted using the E.Z.N.A.Cycle-Pure Kit  . DNA intPRJNA889750).Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform  according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. . The raw reads were deposited into the National Center of Biotechnology Information (NCBI) Sequence Read Archive (SRA) database  with 97% similarity cutoff were clustered using UPARSE version 7.1, and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 against the 16S rRNA database  using a confidence threshold of 0.7.Mothur software (v.1.30.2) was used to calculate the alpha diversity index of OTU level, such as Ace, Chao, and Shannon index, and draw the dilution curve. Beta diversity distance matrix was calculated by Qiime software, and principal co-ordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) diagrams were drawn, respectively. Through linear discriminant analysis effect size (LEfSe) method, the species with significant differences in abundance among different groups were found, and the final difference species were obtained by linear discriminant analysis (LDA). The gene function was predicted and analyzed by PICRUSt2 software, and the relative abundance heat map of functional modules was drawn to find out KEGG metabolic pathway involved by intestinal microbes. All the above drawings were made with R language tools.p < 0.05. Bioinformatic analysis was carried out on Majorbio I-Sanger cloud platform.The experimental data were expressed as mean ± SD. SPSS 23 software was used for statistical analysis, and Graphpad prism 9.0 was used for drawing. After one-way analysis of variance (ANOVA), statistical analysis between groups was performed by Duncan’s multiple range tests, and the differences were considered as significance at p > 0.05). With the progress of the experiment, the food intake, body weight, and blood glucose of the mice in each group showed an increasing trend. The upward trend of mice in the Mod group was higher than all other groups. At week 12 (p < 0.05).Mice fed high-fat diet for 12 weeks to establish NAFLD model. As shown in  week 12 , mice inp < 0.05). The TC level in serum in Mod group was significantly higher comparing with NC group (p < 0.05). However, there were not significantly difference between Mod group and NC group in serum TG level , while AST levels in the Mod group were significantly higher than those in the NC group (p < 0.05). In addition, it can be observed from the histological sections of liver pathology that compared to the NC group (dense structure and neatly arranged hepatocytes), the Mod group of mice had severe lipid accumulation in the liver, the tissue structure was severely disrupted, and the hepatocytes were not arranged in a specific order (p < 0.05).The levels of AST and ALT in serum are important indicators to determine the extent of NAFLD development in mice. In our study , there wic order . After ip < 0.05). However, the levels of leptin and LDL-c were significantly lower and adiponectin in serum was significantly higher in the CaM group compared with Mod group (p < 0.05). There was not significantly difference in the serum HDL-c level between CaM group and Mod group . Contrary to expectations, it was found that camel milk and silymarin interventions did not reduce the degree of impairment of insulin tolerance in mice by calculating the area under the ITT curve (p < 0.05). And camel milk and silymarin regulated the glucose tolerance of the mice to different degrees . After 12 weeks of camel milk and silymarin intervention, the Ace index and Chao index were significantly higher in the CaM group and PC group compared to the Mod group (p < 0.05); the Ace index and Chao index were not significantly different between the CoM and Mod groups (p > 0.05). In summary, camel milk and silymarin can enhance the diversity of intestinal flora Alpha in NAFLD mice.To evaluate the sequencing quality and depth of the 16S rRNA gene in the colonic contents of NAFLD mice, Sob index and Shannon index dilution curves were plotted. The Sob index curve gradually increased with the increase of sample sequencing depth and finally leveled off. And the Shannon index curve reached a plateau . The seqA total of 321 OTUs were detected by sequencing . Long-tep < 0.05), indicating that the structure of intestinal flora has been changed in Mod group observably. The CaM group and the Mod group were distributed in different areas and far away from each other, indicating that the composition of the intestinal microbial community differed greatly between these two groups. The distribution areas of samples in the CoM, CaM, and PC groups partially overlapped, which may be due to the presence of the same or similar microbial communities in these three groups. However, the composition of the gut microbial community in the CoM and PC groups was significantly different from that in the Mod group (p < 0.05). This result infers that camel milk, cow milk, and silymarin can effectively change the structure of intestinal microbial community composition in mice. Furthermore, the results of NMDS plot analysis were highly similar to PCoA plot analysis, which further demonstrated that camel milk had a good regulatory effect on the intestinal microbial community structure of NAFLD mice.To understand the overall differences in intestinal flora diversity between different groups, PCoA and NMDS plots were drawn based on the Brary-Curtis distance algorithm . SamplesThe community Bar map was drawn to understand the dominant species of each group and their proportion. As Faecalibaculum, Bifidobacterium, Bacteroides, Lactobacillus, and an increase in the relative abundance of Dubosiella and unclassified_f_Lachnospiraceae,Coriobacteriaceae_UCG-002  scores of various physiological and biochemical indices were conducted . The indp < 0.05). At the level 2 (p > 0.05). The genes’ functional abundance of metabolism-related increased in the CaM group and PC group after camel milk and silymarin intervention. It can be concluded that camel milk and silymarin induce dynamic changes of genes’ functional abundance related to intestinal flora metabolism in NAFLD mice, which reacted on the intestinal flora bacterial diversity.According to the analysis of intestinal flora diversity in NAFLD mice, long-term intake of high-fat diet changed the structure of intestinal flora in mice. The intestinal flora structure of mice was regulated after camel milk and silymarin intervention. The results of various physiological indicators and tissue sections also demonstrated that camel milk and silymarin could effectively alleviate liver lipid accumulation in mice. Identifying the KEGG metabolic pathway intestinal flora involved in combining intestinal flora data. From  level 2 , there wNon-alcoholic fatty liver disease is a chronic metabolic disease with worldwide prevalence, affecting a large number of people worldwide, characterized by obesity and excessive accumulation of lipids in the liver , 2. The p < 0.05). And from the pathological sections, it can be observed that the Mod group mice have more lipid droplet in the liver and the liver tissue structure is severely damaged. This shows the success of the model building. Moreover, by comparing the body weight changes, blood glucose changes, liver lipid accumulation and liver tissue structure of mice in Mod and CaM groups, it can be determined that camel milk has a protective effect on the liver of NAFLD mice.There are more than 100 trillion microorganisms in the human gut , making it one of the most diverse ecosystems . It has p < 0.05). These results, consistent with the insulin resistance index, suggest that camel milk reduced insulin resistance in NAFLD mice. In addition, camel milk reduced the inflammatory response in the NAFLD mice because of decrease of the levels of IL-6 and TNF-α in serum.Serum biochemical indicators are important in the evaluation of NAFLD. In moderate amounts, leptin enhances hepatic TG transport, but excessive leptin increases the level of TG in the liver , 51. Adip < 0.05), but the Ace index and Chao index of mice in CoM group were not significantly different from Mod group (p > 0.05), indicating that camel milk and silymarin could increase the number of species and the relative abundance of intestinal flora of mice. Beta diversity analysis showed that the samples of NC, Mod, CaM, CoM, and PC groups were distributed in different areas, while CoM and PC groups had some overlapping areas with Mod group; indicating that camel milk had significant effects on the intestinal flora structure of mice, while the effects of cow milk and silymarin on the intestinal flora structure of mice were different from camel milk. Combined with the analysis of physiological and biochemical indicators in mice, we can conclude that camel milk has a positive effect on the regulation of intestinal flora in NAFLD mice.Long-term intake of high-fat diets did not change the Ace and Chao indices of intestinal flora Alpha diversity in the Mod group comparing with the NC group, suggesting that long-term intake of high-fat diets failed to change intestinal flora diversity in mice. We speculate that this may be a characteristic of the feed . Which is different from previous studies, and the exact reason needs to be verified in subsequent supplementary experiments , 56. Aftp < 0.05), indicating that camel milk improved the main components of the intestinal flora of NAFLD mice and had a regulation of intestinal flora diversity in NAFLD mice, thus alleviating NAFLD in mice. This change was also observed in the CoM and PC groups, but unlike the CaM group, NAFLD was not alleviated in the CoM group mice. The results of this study are similar to those of Wang and Zhao et al. . Subsequent clustering analysis by genus and physiological indicators revealed that Bacteroides was significantly and negatively correlated with serum TG level and liver TG level (p < 0.05). This suggests that Bacteroides plays an important role in alleviating hepatic lipid accumulation in NAFLD mice. Short-chain fatty acids in the gut help protect the integrity of the intestinal barrier, and some studies have claimed that norank_f_Muribaculaceae is positively correlated with intestinal short-chain fatty acid levels . After gavage of camel milk, cow milk, and silymarin in mice, the relative abundance of Dubosiella was reduced in the CaM, CoM, and PC groups, with the greatest reduction in the relative abundance of Dubosiella in the CaM group. This indicates that camel milk enhances hepatic TG metabolism and reduces hepatic lipid accumulation, which is consistent with the physiological index, which is also similar to the findings of Bai et al. . These results were consistent with physiological and biochemical parameters and pathological sections of the liver. Studies have confirmed that Alloprevotella has a positive effect on improving the structure of the intestinal flora and protects the integrity of the intestinal barrier , and a significant negative correlation was found between Alloprevotella and the levels of TG in serum and liver, which may be an important reason for the reduced degree of liver lipid accumulation in the CaM group of mice. Lactobacillus and Bifidobacterium are considered to be beneficial bacteria, and the relative abundance of Faecalibaculum is was positively correlated with health status . In conclusion, camel milk, and silymarin reduced the relative abundance of harmful bacteria in the intestinal flora of NAFLD mice, improved the structure of the intestinal flora and protected the integrity of the gut barrier. Although cow milk also changed the structure of intestinal flora in NAFLD mice, it did not effectively prevent lipid accumulation in the liver of mice.The occurrence of disease often means the change of intestinal flora, so the degree of disease occurrence and the trend of improvement can be judged by the structure of intestinal flora. At the phylum level, the ratio of Firmicutes to Bacteroidota affects the major components of the intestinal flora and is important for the assessment of metabolic disorders , 58. In o et al. , 59. At i et al.  demonstrs et al.  results l et al.  also demd levels , 64. In the body , 66. In i et al. . In addi barrier . In the h status , 58. HowCarbohydrate, amino acid, and lipid, as the three basic nutrients of the body, are the basis for the normal physiological activities of the body , 70. CofConclusively, camel milk improved various physiological and biochemical indicators in NAFLD mice, positively regulated the structure of intestinal flora and modulate metabolism-related functional genes. Therefore, the results of this study provide a basis for camel milk to alleviate NAFLD.Dubosiella and Coriobacteriaceae_UCG-002, which were positively correlated with liver TG level, and increased the relative abundance of Bacteroides, norank_f_Muribaculaceae, and Alloprevotella, which were negatively correlated with liver TG level. In addition, camel milk may also enhance the metabolism of the three major nutrients in the organism of NAFLD mice. Therefore, camel milk has a palliative effect on NAFLD in mice.Non-alcoholic fatty liver disease is a metabolic disorder with a wide range of effects, most notably characterized by the excessive accumulation of lipids in the liver. Interventions for NAFLD are particularly important in the absence of drugs specifically for the treatment of NAFLD. Our study demonstrates that camel milk reduced lipid accumulation in the liver, maintained normal liver tissue structure, enhanced hepatic glycolipid metabolism, and reduced inflammatory response in NAFLD mice. The analysis of intestinal flora of NAFLD mice demonstrated that camel milk regulated the intestinal flora structure of mice, increased intestinal flora diversity, reduced the relative abundance of https://www.ncbi.nlm.nih.gov/, PRJNA889750.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by the Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, China.SH, LM, and YL designed the experiments. SH, YL, HL, and LL conducted most of the experiments. SH wrote and edited the manuscript. TJ and RJ reviewed the manuscript. All authors read and approved the manuscript.


Studies on differences in brain function activity between the first depressive episode (FDE) and recurrent depressive episodes (RDE) are scarce. In this study, we used regional homogeneity (ReHo) and amplitude of low-frequency fluctuations (ALFF) as indices of abnormal brain function activity. We aimed to determine the differences in these indices between patients with FDE and those with RDE, and to investigate the correlation between areas of abnormal brain function and clinical symptoms.A total of 29 patients with RDE, 28 patients with FDE, and 29 healthy controls (HCs) who underwent resting-state functional magnetic resonance imaging were included in this study. The ReHo and ALFF measurements were used for image analysis and further analysis of the correlation between different brain regions and clinical symptoms.P < 0.05). Abnormal areas in the temporal and occipital lobes were negatively correlated with the severity of depressive symptoms (P < 0.05).Analysis of variance showed significant differences among the three groups in ReHo and ALFF in the frontal, parietal, temporal, and occipital lobes. ReHo was higher in the right inferior frontal triangular gyrus and lower in the left inferior temporal gyrus in the RDE group than in the FDE group. Meanwhile, ALFF was higher in the right inferior frontal triangular gyrus, left anterior cingulate gyrus, orbital part of the left middle frontal gyrus, orbital part of the left superior frontal gyrus, and right angular gyrus, but was lower in the right lingual gyrus in the RDE group than in the FDE group. ReHo and ALFF were lower in the left angular gyrus in the RDE and FDE groups than in the HC group. Pearson correlation analysis showed a positive correlation between the ReHo and ALFF values in these abnormal areas in the frontal lobe and the severity of depressive symptoms (The RDE and FDE groups had abnormal neural function activity in some of the same brain regions. ReHo and ALFF were more widely distributed in different brain regions and had more complex neuropathological mechanisms in the RDE group than in the FDE group, especially in the right inferior frontal triangular gyrus of the frontal lobe. Major depressive disorder (MDD) is a common clinical psychiatric disorder characterized by affective, cognitive, and somatic symptoms. Most of its clinical manifestations are common symptoms such as depressed mood, diminished interest, slowed thinking, loss of appetite, and insomnia . The WorAccording to the classification criteria of the 10th revision of the International Statistical Classification of Diseases and Related Health Problems, MDD is classified into the first depressive episode (FDE) and recurrent depressive episodes (RDE) . PreviouIn recent years, with rapid developments in neuroimaging technology, resting-state functional magnetic resonance imaging (rs-fMRI) has gradually been applied to the study of insomnia , schizopHowever, the combination of ALFF and ReHo has been mainly applied in studies on mild cognitive impairment , schizopIn this study, we aimed to determine the differences in brain function activity among patients with FDE, patients with RDE, and healthy controls (HCs) by using the ReHo and ALFF methods based on rs-fMRI techniques. Further, we also aimed to analyze the correlation between different brain areas and clinical characteristics. We hypothesized that differences would be observed in the brain neural circuits of patients with FDE and those with RDE, especially in areas closely related to the frontal lobe. This study will provide a neuroimaging basis for the differences in neuropathological mechanisms between FDE and RDE and some insights for clinical research.n = 31; mean frequency of recurrence, 2.27 (standard deviation = 0.79)] and those with FDE . The inclusion criteria were (1) age 18–55 years; (2) HAMD-17 score > 17; and (3) The FDE group all had their FDE prior to enrollment and were not receiving any antidepressant medication. The RDE group had a previous history of depression, cured by antidepressant medication, now recurring and a history of antidepressant withdrawal for at least 4 weeks prior to enrollment. We also included 29 sex- and age-matched HCs (21 women and 8 men) who (1) were aged 18–55 years, (2) had a HAMD–17 score of < 7, (3) had right-handedness, and (4) had no history of any mental illness in first-degree relatives.A total of 59 outpatients with MDD from Guang’anmen Hospital, Chinese Academy of Traditional Chinese Medicine, Beijing First Hospital of Integrative Medicine, were recruited for this study. All patients met the Diagnostic and Statistical Manual of Mental Disorders Fifth Edition criteria for MDD. We used the 17-item Hamilton Rating Scale for Depression (HAMD-17)  to assesThe exclusion criteria for patients and HCs were as follows: (1) serious mental illness and other diseases such as cardiovascular and cerebrovascular disorders; (2) history of drug and alcohol abuse; (3) any contraindications to MRI, such as presence of a heart pacemaker, metal fixed false teeth, or severe claustrophobia; (4) pregnant or lactating status; and (5) bipolar disorder or suicidal ideation.All patients were required to sign an informed consent form before enrollment. This study was approved by the ethics committee of Guang’anmen Hospital, Chinese Academy of Traditional Chinese Medicine.All patients in this study underwent MRI using a Magnetom Skyra 3.0-T scanner . Before the scanning procedure, the patients were instructed to remain awake and avoid active thinking. During the scanning process, the patients were required to wear earplugs and noise-canceling headphones, to use a hood to immobilize the head, and to lie flat on the examination bed. The scanning procedure involved a localizer scan, high-resolution three-dimensional T1-weighted imaging, and BOLD-fMRI.The scanning parameters were as follows: for three-dimensional T1-weighted imaging, time repetition/time echo = 2,500/2.98 ms, flip angle = 7°, matrix = 64 × 64, field of view = 256 mm × 256 mm, slice thickness = 1 mm, slice number = 48, slices = 192, scanning time = 6 min 3 s; for BOLD-fMRI, time repetition/time echo = 2,000/30 ms, flip angle = 90°, matrix = 64 × 64, field of view = 240 mm × 240 mm, slice number = 43, slice thickness/spacing = 3.0/1.0 mm, number of obtained volumes = 200, and scanning time = 6 min 40 s.1 (The rs-fMRI data were preprocessed using DPARSF (Data Processing Assistant for rs-fMRI) software (DPABI5.0)1  in MATLAThe DPARSF  software was used to analyze the ReHo and ALFF of the pre-processed data. The similarity of the time series of each voxel to its neighboring voxels (26 neighboring voxels) was assessed using the Kendall’s coefficient of concordance (KCC) , i.e., Rz transformation before statistical analysis.Data were spatially normalized and smoothed, and a fast Fourier transform was performed to switch the time series to the frequency domain to obtain the power spectrum. The square root of the power spectrum at each frequency was calculated to obtain the average square root of the ALFF measurement for each voxel in the range of 0.01–0.08 Hz. Finally, time bandpass filtering (0.01–0.08 Hz) was performed. To reduce the inter-individual variability, ALFF was transformed to zALFF using Fisher’s t-test was used to compare the duration of disease, HAMD-17 scores, and frequency of recurrence between the two patient groups, with a threshold of P < 0.05 (two-tailed) set as statistically significant.Clinical data were analyzed using the SPSS 23.0 statistical software . One-way analysis of variance was used to compare age and educational level among the three groups, and the chi-square test was used to compare sex differences. A two-sample P < 0.05 (two-tailed), and threshold voxel levels of P < 0.005 were defined as statistically different. We performed post-hoc t-test analysis using DPARSF 5.1 software for two-by-two comparisons between groups, and Bonferroni correction was applied to the results, setting a threshold of P < 0.016 (0.05/3) for statistical significance. The threshold was set to clusters > 10 voxels.Imaging data were analyzed using the DPARSF toolbox, and a voxel-based one-way analysis of variance was performed to compare the whole-brain ReHo/ALFF map among the three groups. Sex, age, educational level, and framewise displacement (a metric derived from Jenkinson’s formula) were used as covariates, and brain areas with ReHo/ALFF differences among the three groups were corrected for Gaussian random fields. The corrected cluster level was set at P < 0.05 (two-tailed).To verify the relationship between ReHo/ALFF values and clinical symptoms, we extracted the mean ReHo/ALFF values of three different brain regions and performed Pearson correlation analysis of the clinical scale scores of each group. Significance was set at a statistical threshold of Two patients with RDE were excluded because of excessive head movement displacement. Therefore, a total of 29 patients with RDE, 28 patients with FDE, and 29 HCs met the inclusion criteria. No statistical differences among the three groups were found in terms of sex, age, and years of education. The HAMD-17 scores were not statistically different between the RDE and FDE groups, whereas a statistical difference was observed in the duration of illness and frequency of recurrence .One-way analysis of variance showed significant differences in ReHo and ALFF among the three groups in the right inferior frontal triangular gyrus, left anterior cingulate cortex, left middle temporal gyrus/left angular gyrus, and left inferior temporal gyrus/left inferior occipital gyrus. Meanwhile, ReHo was significantly different in the left superior temporal gyrus and right Rolandic operculum gyrus. ALFF was also significantly different in the right angular gyrus, right lingual gyrus, and Vermis_3 , 2.Compared with the FDE group, the RDE group had elevated ReHo in the right inferior frontal triangular gyrus and decreased ReHo in the left inferior temporal gyrus. Meanwhile, the RDE group had elevated ALFF in the right inferior frontal triangular gyrus, left anterior cingulate gyrus, orbital part of the left middle frontal gyrus, orbital part of the left superior frontal gyrus, and right angular gyrus, but had decreased ALFF in the right lingual gyrus .Compared with the HC group, the RDE group had elevated ReHo in the right inferior frontal triangular gyrus, left anterior cingulate gyrus, and left superior temporal gyrus, but had reduced ReHo in the left angular gyrus. Meanwhile, the RDE group had elevated ALFF in the left anterior cingulate gyrus and right angular gyrus, but had decreased ALFF in the left angular gyrus .Compared with the HC group, the FDE group had elevated ReHo in the left inferior temporal gyrus and decreased ReHo in the left middle temporal gyrus/left angular gyrus. Meanwhile, the FDE group had elevated ALFF in the left inferior temporal gyrus and Vermis_3, but had decreased ALFF in the left middle temporal gyrus/left angular gyrus .r = 0.436, P = 0.018; r = 0.394, P = 0.034). Meanwhile, we observed a positive correlation between the ReHo values in the left anterior cingulate cortex/orbital part of the left middle frontal gyrus and the HAMD-17 scores in the FDE group . Furthermore, we found a negative correlation between the ReHo values in the left inferior temporal gyrus/left inferior occipital gyrus and right lingual gyrus and the HAMD-17 scores in the FDE group   .To our knowledge, this is the first study to investigate the changes in local brain function activity in patients with RDE or FDE using the ReHo and ALFF methods. The results of this study showed that the RDE and FDE groups had abnormal neural function activity in some of the same brain regions. ReHo and ALFF were more widely distributed in different brain regions and had more complex neuropathological mechanisms in the RDE group than in the FDE group. This study provides a reference for the differences in brain function activity between RDE and FDE.We found increased ReHo and ALFF in the right inferior frontal triangular gyrus in the RDE group compared with the FDE group. The right inferior frontal triangular gyrus is located in the dorsolateral prefrontal cortex (DLPFC), an important component of executive function that is closely related to working memory, thought activity, and cognitive control . PreviouWe also found differences in ALFF in the left anterior cingulate gyrus and left orbitofrontal gyrus between the RDE and FDE groups. The anterior cingulate gyrus is an important component of the limbic system. It has extensive fibrous connections to many cortical and subcortical structures; is involved in the regulation of a wide range of functions, such as emotion, cognition, and motivation; and is closely associated with the onset of depression . PreviouIn addition, we found that the RDE group had higher ALFF values in the right lingual gyrus than the FDE group. The lingual gyrus is part of the occipital lobe, and previous studies have shown that the lingual gyrus is also involved in activities related to visual memory processing and is closely associated with the development of MDD . One stuMeanwhile, we found that the ALFF in the right angular gyrus was higher in the RDE group than in the FDE group. A previous study reported that the right angular gyrus is associated with the self-localization function in humans and is potentially associated with psychiatric disorders, and that an abnormality in this function affects both sensory and perceptual functions, with a causal relationship between them . This maInterestingly, we also found that the RDE and FDE groups had lower ReHo and ALFF in the left angular gyrus than the HC group. The angular gyrus is located in the posterior part of the inferior parietal lobe and is mainly involved in human semantic and numerical processing, memory retrieval, spatial cognition, word reading and comprehension, reasoning, and social cognition, with the left angular gyrus playing a more important role in situational simulation and memory . The angSome limitations of this study should be considered. First, the patients with RDE enrolled in this study might have still been affected by an underlying antidepressant action despite having stopped medication for 4 weeks. Second, the number of recurrences in the RDE group was inconsistent, and a first-recurrence study of patients with RDE seem to have greater research value. Third, this study focused on only one scale (HAMD-17). To enhance the scientific value of this study, more scales need to be used in the future to focus on the detailed correlation of cognitive, somatic, anxiety, and insomnia symptoms with RDE and FDE. Finally, this study did not find a difference between RDE and FDE in depressive symptoms, which may be related to the specificity and small size of the study population. Further studies with a larger sample size are needed to confirm or refute the findings of this study.This study used ReHo and ALFF, which are indices based on the rs-fMRI technique, to preliminarily analyze the differences between RDE and FDE in terms of neural activity in different brain regions. The RDE and FDE groups showed abnormal changes in neural function activity in some of the same brain regions, with ReHo and ALFF being more widely distributed in different brain regions and the neuropathological mechanisms being more complex in the RDE group than in the FDE group, particularly in the right inferior frontal triangular gyrus of the frontal lobe.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.China Clinical Trials Registry, chiCTR1800014277. The patients/participants provided their written informed consent to participate in this study.The experimental protocol was approved by the Ethics Committee of Guang’anmen Hospital, Chinese Academy of Traditional Chinese Medicine (No. 2017-021-SQ), Trial registration: JF conceived and designed this experiment. JS wrote and revised the manuscript and participated in the collection of cases and statistical analysis of the data. LC drew diagrams and made statistical analysis of data and revised the manuscript. ZD analyzed the data and revised the manuscript. JH, DG, YM, ZW, CG, and YL participated in data analysis, case collection, and manuscript writing. YH, LZ, and JC performed fMRI on the patient. FX, XH, XX, JT, and XY involved in case collection and symptom assessment of patients. 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.


Diurnal temperature range (DTR) has been increasingly recognized as a risk factor for mortality and morbidity, but the association between DTR and acute lower respiratory infection (ALRI) outpatient visits has not been examined among children in China.A total of 79,416 ALRI outpatient visits among children were obtained from the Guangdong Second Provincial General Hospital between 2013 and 2019. DTR was calculated by taking the difference between the maximum and the minimum temperatures. Generalized additive models using a quasi-Poisson distribution were used to model the relationship between DTR and ALRI outpatient visits.Diurnal temperature range was significantly associated with elevated risks of ALRI outpatient visits: the excess risks (ERs) and 95% confidence intervals (CIs) were 2.31%  for ALRI, 3.19%  for pneumonia, and 1.79%  for bronchiolitis, respectively. Subgroup analyses suggested that the associations were significantly stronger during rainy seasons  than those in dry seasons , while no significant effect modifications were found in sex and age groups.Diurnal temperature range may elevate the risk of ALRI outpatient visits among children in China, especially during rainy seasons. Public health policies are needed to mitigate the adverse health impacts of DTR on children. Lower respiratory tract infection primarily includes pneumonia and bronchiolitis, and it is the leading cause of under-five mortality across the globe , 2. A coClimate change has been recognized as the single biggest threat to human health , 9. The In this time-series analysis of acute lower respiratory infections outpatient visits in a large tertiary hospital in Guangzhou, China, we propose to quantify the association between DTR and the outpatient visits of ALRI. We hypothesize that DTR can elevate the incidence of outpatient visits with ALRI.Guangzhou, in Southern China, belongs to a subtropical humid-monsoon climate, and the annual mean temperature is 22°C and average rainfall is 1,500–2,000 mm, with 18.6 million people in 2020. Guangzhou city has a maritime monsoon climate with an average annual temperature of 22°C, and an average relative humidity of 80%. As the capital city of Guangdong Province, the large number of residents yielded sufficient statistical power and long panels of observation, and the health outcome data have higher quality .The data about ALRI outpatient visits from February 2013 to December 2019 were obtained from the Guangdong Second Provincial General Hospital, which is one of the largest tertiary hospitals in Guangzhou, China . This adhttp://data.cma.cn/), including daily maximum, mean, and minimum temperature, and relative humidity. Daily meteorological data across the Guangzhou station were used to reflect the general population's daily exposure. The meteorological data were collected from national regular monitoring stations and there were no missing data. Following the definitions of several previous studies , PM10, nitrogen dioxide (NO2), and sulfur dioxide (SO2) in Guangzhou during the study period  is the expected daily number of ALRI on day t, β means the coefficient of the DTR, s represents a smoothing function, t is the adjustment for long-term and seasonal trends, Temp03 is the moving average for the previous 4 days' temperature, DOW is an indicator for the day of the week, RH presents the humidity on the current day, and PH represents a binary variable for the public holiday.where E up to seven days lag (lag7). In multi-day lags models, we considered the accumulated effects .To examine whether the adverse impacts of DTR on ALRI differed by sex , age group (age <5 vs. age 5–14), and season (dry vs. rainy), we conducted subgroup analyses stratified by these factors. Due to the tropical monsoon climate, the dry season was defined as the period from November to April of the following year, and the rainy season was from May to October. We estimated whether the differences between groups were significant by calculating the 95% CI as:1 and E2 are the estimates for the two groups, and SE1 and SE2 are their corresponding standard errors  , 31. Secp-value < 0.05 was considered the statistically significant.The results in this study were reported as excess relative risk (ER) and 95% CI. All the statistical analyses were conducted in R version 4.1.0 . A p-val2.5, PM10, NO2, and SO2). The matrix of pairwise Pearson correlation coefficients between the environmental variables are shown in To further understand potential non-linear relationships between DTR and risk of ALRI (pneumonia and bronchiolitis) outpatient visits, we constructed non-linear spline models to estimate the concentration-response curves for this relationship. We conducted a set of sensitivity analyses to check the robustness of the main results. First, we tested the influence of the dfs for temporal trends and meteorological variables on the main results by alternating them from five to eight. The results  suggest We further examined the associations between DTR and ALRI outpatient visits by sex, age group (<5 years and 5 to 14 years), and seasons to test for potential effect modification. In this large time-series study of 79,416 ALRI outpatient visits spanning seven years in Guangzhou, China, we found that higher DTR was associated with significantly higher risks of outpatient visits of ALRI, pneumonia, and bronchiolitis. The results were consistent when alternative dfs were applied to temperature and relative humidity, as well as daily concentrations of air pollution were adjusted for. Further spline analyses suggested close-to-linear relationships between DTR and ALRI outpatient visits. We also found significant effect modification by season, where the ERs were significantly larger during rainy seasons than those in dry seasons.Numerous studies have investigated the associations of temperature and its variability with the incidence of respiratory diseases –36. For via similar pathways (Although this time-series epidemiological study does not illustrate the physiological mechanisms underlying associations between DTR and ALRI, several biological mechanisms could underpin our observed relationships. Dramatic temperature change can afflict the circulation, immune, and thermoregulatory system, causing abnormality of several laboratory indicators including heart rate, blood pressure, blood viscosity, and oxygen levels , 37–39. pathways .The effect modification of the associations between DTR and respiratory diseases by season is also reported by several previous studies , 41, 42.Although DTR in China showed a slightly decreasing trend in the recent few decades , 45, theA few limitations should be noted for this study. First, this is a time-series analysis of daily counts of outpatient visits and the results are subject to potential ecological fallacy bias . Second,Our study included complete data on ALRI outpatient visits spanning a consecutive 8 years from a large tertiary hospital, and the fair amount of sample yielded large statistical power and generated significant model results. The public health implications of this study are that the parents or guardians of children need to be aware of temperature change and adopt effective measures to minimize the adverse health effects of DTR on the occurrence of respiratory diseases among children.In summary, this multi-year time-series study suggests that DTR may increase the outpatient visits of ALRI, pneumonia, and bronchiolitis among children. The effects of DTR may be stronger during rainy seasons. Relevant health policies, including health education and promotion, are needed to mitigate the deleterious health impacts.zhangxiaogd2h@163.com.The data analyzed in this study is subject to the following licenses/restrictions: We do not have permission to share data. Requests to access these datasets should be directed to XZ: ZL and XZ: investigation and visualization. QM, ZL, and XZ: supervision. ZZ: writing—original draft. ZZ, DX, JC, and QM: writing—review and editing. 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.