Posttraumatic stress disorder (PTSD) may develop as a serious long-term consequence of traumatic experiences, even many years after trauma exposure. Dessie town residents have experienced prolonged armed conflict due to inter-communal conflict in 2021. Those people are exposed to different kinds of trauma, and violence, making them more prone to psychological disorders. Despite the highest number of people affected due to conflict and its negative impact on mental health, post-traumatic stress disorders among people are overlooked in Ethiopia. This study aimed to assess the prevalence and associated factors of post-traumatic stress disorder among people who experienced traumatic events in Dessie town, Ethiopia, 2022.P-values less than 0.05 was considered statistically significant.Community based cross-sectional study was conducted on June 8–July 7, 2022, by using a multi-stage cluster sampling with a total sample of 785. Pretested, structured questionnaires and face-to-face interviews were used for data collection. PTSD was assessed by the Post-traumatic stress disorder Checklist for DSM-5 (PCL-5). Data was entered using Epi-data version 3.1 and, then exported to SPSS version 26 for analysis. The association between outcome and independent variables was analyzed with bi-variable and multivariable logistic regression. The prevalence of PTSD among Dessie town residents was found to be 19.4% . In multivariable analysis, being females , previous history of mental illness  depressive symptoms , witnessing a serious physical injury of a family member or friend  and high perceived life threats  were found to be significant predictors of PTSD.The prevalence of PTSD among Dessie town residents was found to be huge. Being female, having a previous history of mental illness, depressive symptoms, witnessing a serious physical injury of a family member or friend, and high perceived life threats were variables that are independent predictors of PSTD. People who have experienced such a severe traumatic event require psychosocial support to aid in their recovery from the terrible experiences. According to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders DSM-5), Posttraumatic stress disorder (PTSD) is one of the mental disorder related to the following exposure to a traumatic or stressful event which is characterized by the symptoms of intrusion, avoidance, alternation of mood and cognition and hyperarousal lasts for more than a month after the stressful event . Experie, PosttraIn post-conflict and conflict-ridden societies, the prevalence rates in the general population can be much higher . AccordiA meta-analysis study conducted on a global population of adult war survivors that looked at all countries that suffered at least one war within their territory between 1989 and 2015 found that 23.81% of adult war survivors met the diagnostic criteria for PTSD . AnotherMany studies have shown a link between PTSD and various risk factors before the injury like sex, low educational status, low social support, unemployment, lower socioeconomic status, younger age, childhood abuse, history of depressive symptoms, and history of mental illnesses –25. AlsoConflicts reportedly killed over three times more people during the past decade than natural disasters . In EthiA community-based cross-sectional study was conducted in Dessie Town, Northeast Ethiopia, from June 8 to July 7/2022. The administrative center of Dessie Town in the Amhara Region; is located from Addis Ababa to Northeast Ethiopia 401 Km. It sits at a latitude and longitude of 11°8′N 39°38′E, with an elevation between 2,470 and 2,550 meters above sea level. It has 18 kebeles and has a 350,000 population. Among those populations 186,571 males and 163,429 females according to 2016 to 2017 South Wollo Zone statistics office data. The two governmental hospitals have psychiatric outpatient and inpatient services.All residents of Dessie town, North East, Ethiopia.Households live in the Menafesha sub-city who are in the selected kebele and available during the study period.All households in selected kebeles and one individual from each household who was 18 years old and above, living in Dessie town during the time of the study were included, while participants who were not available during wartime, severely ill, and unable to communicate during data collection time and those residents less than 6 months were excluded from the study.P-value of 58.4% from the previous study in Southern Ethiopia .where: n = sample size, P = prevalence of previous study (58.4%), d = degree of precision (assumed to be 5%), (z α/2)Since we have employed a multi-stage sampling technique to consider the designing effect, the calculated sample size by two to correct the sampling error. After all, by adding a 10% non-response rate, the final sample size was 821.j = Nj/nj where; Kj = sampling interval, Nj = total number of households, nj = total number of sample size. There are three kebeles within the Menafesha sub-city and having a total of 3,832 households, K or interval was decided by N/n (K = 3832/821 = 4). They interviewed one individual from every four households consecutively  with scores ranging from 0 to 80 with a five-point Likert scale . A score of ≥ 33 was considered a symptom of PTSD . The PCLThe Patient Health Questionnaire (PHQ-9) was used to assess depression, with a score of 10 or higher indicating depression . In seveThe Social Support Scale (The Oslo three-items) (OSSS-3) was used to collect data regarding the strength of social support. It was categorized into three broad categories of social support; 3–8 poor social support, 9–11 moderate social support, and 12–14 strong social support . The intn = 41) of the participants took part in the pre-test, which aimed to identify any potential issues with the data collection methods and suggest changes to the questionnaire. The principal investigator and supervisor regularly oversaw and assisted the data collectors. Every day during the period of data collection, supervisors and primary investigators verified the data for consistency and completeness.The questionnaire was initially written in English, then translated into Amharic, and finally back into English to ensure uniformity. Additionally, we use standard tools to determine the outcome variable by giving 2 days of training for data collectors and supervisors. In the Hotie sub-city, 5%  of the participants was 36.01 (± 11.29 years), and the majority of 447 (56.9%) were males. The majority of the participants, 521 (66.4%), 519 (66.1%), and 203 (25.9%) were married, Muslim, and had a University degree and above, respectively. The majority of the community were 249 (31.7%) merchants by occupation. According to the participant’s responses, 460 (58.6%) of respondents earned monthly income ≥ 2166 (ETB) .According to the current study, 40 (5.1%) of respondents had a history of mental illness. Among participants, 39 (5.0%) respondents had a family history of mental illness and 88 (11.2%) participants reported a history of chronic medical illness. Of the participants, 192 (24.5%), and 262 (33.4%) had depressive symptoms and anxiety symptoms, respectively .Regarding the use of the substance, nearly one-fourth 186 (23.7%) had consumed alcohol, 196 (25%) had used khat, and 8.7% had smoked cigarettes in their lifetime. Whereas, 125 (15.9%) consumed alcohol, 134 (17.1%) use khat, and 43 (5.5%) smoke cigarette currently .Regarding individual trauma type, the type of trauma experienced by the community 72 (9.2%), 33 (4.2%), 66 (8.4%), and 116 (14.8%) were serious physical injury during the conflict, witness the death of a family member, witness a serious physical injury of a family member or friend, and property destroyed during the conflict, respectively. From this study, 120 (15.3%) had experienced childhood abuse, 357 (45.5%), had moderate perceived life threats and 420 (53.5%) had received threat intermittent social support .In this study, the overall prevalence of PTSD among people who experienced traumatic events in Dessie town was 19.4% .P–value of < 0.25 and became a candidate for multivariable analysis. Variables such as, being female, history of mental illness, depressive symptoms, witnessing a serious physical injury of a family member or friend, and high perceived life threat were found to be significantly associated with PTSD at a p-value less than 0.05. in multivariable analysis.In the bivariate analysis, age, sex, history of mental illness, family history of mental illness, history of chronic medical illness, childhood abuse history, ever alcohol use, current alcohol use, serious physical injury during the conflict, witness the death of a family member, witness a serious physical injury of a family member or friend, the property be destroyed during the conflict, depressive symptoms, perceived life threat, and low social support showed a Females were 1.63 times more likely to develop PTSD than males . Those participants who had a previous history of mental illness were 3.14 times more likely to develop PTSD as compared with respondents who did not have a history of mental illness . Likewise, the odds of developing PTSD among those with depressive symptoms were 3.12 times higher as compared to their counterparts . Those who had witnessed a serious physical injury of a family member or friend were 2.82 times more likely to develop PTSD than those who had not witnessed a serious physical injury of a family member or friend . Finally, the odds of developing PTSD among participants who had experienced high perceived life threats were five times higher than those who had low perceived life threats  .War is complex and potentially leads to ever-lasting trauma in the population . This poThe result of the current study indicated that the magnitude of PTSD among people who experienced traumatic events in Dessie town was 19.4% . The finding was congruent with the study carried out among traumatic Patients in West Ethiopia 17.1% , Serbia .7, 22.0]This result was higher than those found in studies in Uganda 11.8% , SoutherRegarding the associated factors, females were 1.63 times more likely to develop PTSD than males. This finding was in agreement with different studies in South Africa , and twoIn the current study, we found that participants who had a previous history of mental illness were 3.14 times more likely to develop PTSD as compared to those who did not have a history of mental illness. Similar to a finding of different studies from Kenya , and SouThe odds of developing PTSD among those with depressive symptoms were 3.12 times higher as compared to their counterparts. This is similar to findings done in Kenya , MaikadrThis finding also revealed that participants who had witnessed a serious physical injury of a family member or friend were nearly three times more likely to develop PTSD than those who had not witnessed a serious physical injury of a family member or friend. This was supported by the study conducted in Bangladesh , and theFinally, the odds of developing PTSD among participants who had experienced high perceived life threats were five times higher than those who had low perceived life threats. A current study finding was congruent with a finding from North West, Ethiopia 59.8% , Koshe lThe use of a high response rate and the inclusion of significant variables that were left out of earlier research. Also, for measuring PTSD, we employed an updated standardized tool even it is not validated in Ethiopia. Additionally, to evaluate independent variables including depressive symptoms and perceived stress, validated and standardized instruments were used. Whereas; we were unable to conclude the causes of the connections we discovered because of the study’s cross-sectional nature. The research could not include the afflicted demographic as children in traumatic events, this is recommended for future researchers to perform their studies among this affected population. In addition, recall bias and social desirability may potentially be further limitations. Because the data was collected through a face-to-face interview, which could influence participants’ responses to socially acceptable questions about substances, people with PTSD symptoms may be more motivated to remember earlier exposure than those without the symptoms.The current study showed a high prevalence of PTSD among people who experienced traumatic events in Dessie town. Being female, a previous history of mental illness, depressive symptoms, witnessing a serious physical injury of a family member or friend, and high perceived life threats were found to be significant predictors of PTSD. Therefore, mental health programs by local officials, psychologists, and non-governmental groups should be expanded for screening and providing treatment for all people suffering from PTSD to minimize the prevalence of this condition. In addition, mental health awareness campaigns for trauma victims to seek mental health treatment and intense and persistent psychosocial interventions should be provided.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 Ethical Committee of Wollo University College of Medicine and Health Science with an ethical review board with an ethical review number (RCSPG-205/14). All study participants were told that participation was completely voluntary, that written informed consent was obtained, and that they could withdraw from the study at any time if they were not comfortable. A participant’s confidentiality and privacy were ensured by not including a personal identifier. All methods were performed in accordance with the relevant guidelines and regulations. The patients/participants provided their written informed consent to participate in this study.TA was the principal investigator of the study and involved from inception to design acquisition of data analysis, interpretation, and drafting and preparing of the manuscript. YZ, MS, AA, and NB were involved in the reviewing of the proposal and critical review of the draft manuscript. All authors read and approved the final manuscript.


Approximately 230 million children under 5 years old of age suffer from malnutrition and over half of the children below 5 years old deaths are due to malnutrition nowadays. To gain a better understanding of this problem, the application of spatial analysis has risen exponentially in recent years. In this review, the present state of information on the use of spatial analysis in childhood malnutrition studies was evaluated using four databases of digital scientific journals: ScienceDirect, Scopus, PubMed and CINAHL. We chose 2,278 articles from the search results and a total of 27 articles met our criteria for review. The following information was extracted from each article: objective of study, study area, types of malnutrition, subject, data sources, computer software packages, spatial analysis and factors associated with childhood malnutrition. A total of 10 spatial analysis methods were reported in the reviewed articles and the Bayesian geoadditive regression model was the most common method applied in childhood malnutrition studies. This review highlights the importance of the application of spatial analysis in determining the geographic distribution of malnutrition cases, hotspot areas and risk factors correlated with childhood malnutrition. It also provides implications for strategic initiatives to eradicate all forms of malnutrition. Malnutrition is a complicated issue that is believed to cause more than one-third of all mortality, despite the fact that it is rarely mentioned as the primary cause . UndernuMalnutrition in children is caused by a variety of factors. Poverty has typically been the leading cause of childhood malnutrition . Other fNowadays, the availability of updated satellite imagery and internet data, as well as the usability of geographic information system (GIS) applications, allows for faster innovation and development . GIS proA review article on the implementation of spatial analysis in childhood malnutrition exploration was carried out by Marx et al. . HoweverThe primary objectives of this review were to: i) analyse the use of spatial analysis methods in the studies of the geographic variation of childhood malnutrition and its potential factors; ii) synthesise the spatial analysis methods of the selected studies and separate the methods into three categories: spatial regression model, spatial autocorrelation/clustering and spatial interpolation and iii) evaluate the benefits and disadvantages of using spatial analysis methods in childhood malnutrition studies. The following questions were posed for this review: i) What spatial analysis methods have been used to analyse childhood malnutrition? ii) What spatial analysis method is frequently used in childhood malnutrition studies? iii) What are the benefits and disadvantages of spatial analysis in childhood malnutrition studies? The findings from this review will make a significant contribution to the production of information that can be used to develop policies that improve children’s nutrition security. Furthermore, insights into strategic interventions to eradicate all forms of childhood malnutrition can be obtained. To make the review coherent, we structured it to contain some background information, an objective, a summary of datasets and eligibility, results, discussion and conclusion.The Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) were utilised for this review . PRISMA-The systematic search of the present study was conducted on four digital scientific journal databases: ScienceDirect, Scopus, PubMed and CINAHL (EBSCO host interface).The following terms were used to identify related articles in conjunction with keyword searches: ‘spatial’ OR ‘geographic information system’ OR ‘mapping’ AND ‘childhood malnutrition’. The terms were paired with the logical OR operator, while the logical AND operator was used to merge the two terms. Only articles conducting spatial analysis methods to discuss childhood malnutrition were identified as relevant to this review.The articles were chosen for evaluation if they met the requirements for inclusion criteria. The first criterion was the literature type, with the researchers deciding to concentrate solely on journals (research articles) because they served as a primary source of empirical data. As a result, a systematic review, review, meta-analysis, meta-synthesis, book series, books, book chapters and conference proceedings were excluded from this study. This review only included research articles published in English between January 2010 and March 2020. Research articles that have not explicitly reported spatial analysis applications in childhood malnutrition studies were removed from this review.The titles, abstracts and contents of all articles were fully reviewed to verify that they met the inclusion criteria and were appropriate for use in the current study to meet the objectives. In the first stage, the selection of articles was carried out independently by three reviewers by screening the titles after the database search. The purpose of this stage was to remove duplicate articles. If either of them decided the title of an article was relevant, the article was included—otherwise, the article was excluded. All selected articles were subjected to abstract screening in the second stage. The reviewers must agree to include an article based on the defined relevance criteria during this stage. Any disagreements were resolved by discussion and consensus among all reviewers. When the abstracts of articles could not be presented as relevant or irrelevant, their full-text articles were retrieved for additional review. The full-text articles that met the inclusion criteria were further evaluated and information was retrieved from each article.Information from each of the selected articles, such as the objective of the study, study area, types of childhood malnutrition, subject of the study, data sources, computer software packages, spatial analysis (method) and factors associated with childhood malnutrition, was extracted using Microsoft Excel. Then, we further distinguished the spatial analysis methods used in the selected articles into the categories of: i) spatial regression model; ii) spatial autocorrelation/clustering and iii) spatial interpolation. The frequency of each spatial analysis used was tabulated in a table. It should be noted that many types of spatial analysis might be applied in a study.n = 19). Thus, a total of 27 relevant articles  were selected in this review. Our initial search in the ScienceDirect database yielded 644 articles, the Scopus database yielded 419 articles, the PubMed database yielded 34 articles and the CINAHL database yielded 1,178 articles. A total of 2,278 articles were listed, of which 1,912 articles were chosen based on research articles. A total of 1,819 articles were removed based on their title (title screening) and 18 duplicates were removed at the screening stage. In the second stage, 75 abstracts of articles were reviewed and only 46 articles were selected for full-text review. After reviewing the full text for each of the articles, we excluded all articles that did not use the spatial analysis method and did not focus on childhood malnutrition issues  had a research objective containing geographical terms such as distribution, spatial or geographical variability, spatial clustering, and regional differences. All the studies selected for this review were conducted in developing countries . More than half of the studies  were conducted in African countries  studied child stunting           , 35, 37,, 33.3%) , 37, 39,, 22.2%) , 39, 40 , 11.1%) , 39, 40..1%) n = , 85.2%  –39, 41, 1, 3.7%) , one stu1, 3.7%) , one stun = 15, 55.6%)           , 40, 41,, 14.8%) , 22, 37,2, 7.4%) , 32, two2, 7.4%) , 34, two2, 7.4%) , 28, one1, 3.7%) , one stu1, 3.7%) , one stu1, 3.7%)  and one 1, 3.7%) .n = 6, 22.2%)      (R-INLA) packages  , WinBUGS , SaTScan , BayesX  and QGIS . Four articles did not mention the computer software packages they used in their studies .Climate data  was used by six studies , 22.2%) , 32, 33,, 14.8%) , 32, 33,, 11.1%) , 29, 32,2, 7.4%) , 32 and 2, 7.4%) , 35. For, 33.3%) , with fopackages . The sec.2% , 32,, 29.6%) , 39, 40,The spatial analysis used in childhood malnutrition studies by the selected articles is presented in n = 11, 40.7%)     , 37, 41., 14.8%) , 22, 33,, 11.1%) , 18, 22 2, 7.4%) , 22.n = 10, 37.0%)      –28, 38. , 18.5%) , 25, 38., 11.1%) , 39, 40,, 11.1%) , 30, 35 2, 7.4%) , 27.n = 2, 7.4%)  , 33. In 2, 7.4%) , the span = 27) implies that spatial analysis is still new to childhood malnutrition studies. However, considering that spatial analysis is still rarely used in the field of childhood malnutrition, this result is not surprising.In our search of more than 10 years (January 2010–March 2020) of research articles on childhood malnutrition studies, we found that only 27 studies used spatial analysis in their studies. This might be connected to the particular search strategy. We aimed to eliminate false positives, as is the norm in systematic reviews, by choosing search words that were sensitive but not specific. The inclusion of all articles resulted in a vast number of impractical studies, with only a few articles appearing to be relevant. Since we may have overlooked a few articles in this review, we concluded that the specificity of the study did not significantly alter our findings. The small number of chosen articles  from the median height-for-age value of the WHO Child Growth Standards in 2013 , 31.In public health research, GIS methods are applied in several ways. Spatial data exploration and visualisation are two of the most function of GIS. This review has demonstrated several spatial analysis methods used in childhood malnutrition studies. The Bayesian geoadditive regression model is the most commonly used to investigate the spatial dimension of childhood malnutrition. This method was predominantly used because it promotes the calculation of the conditional association between the state of childhood malnutrition and spatial dependency within the country and across regions . The finOther methods of spatial regression models, such as OLS, SLM and SEM regression, have also been applied by several studies to deepen knowledge of the spatial aspect of childhood malnutrition. The fundamental principle of SLM is that the results of the dependent variables are influenced in the neighbourhood areas, whereas SEM is used to assess the influence of certain variables that are not present in the regression model but have an impact on the output variables. The primary difference between the two models is that, unlike SEM, SLM does not include spatial dependency in the error term. A study by Khan and Mohanty  found thThe spatial autocorrelation/clustering is a method for determining the existence and absence of non-random spatial patterns in a study area. These approaches, also known as hotspot analysis, yield estimates disaggregated to the spatial unit level to characterise significantly autocorrelated local areas. In this review, this method was used by the selected articles to identify areas where childhood malnutrition cases are higher or lower than expected under the assumption that childhood malnutrition cases are uniformly distributed spatially. If the cases are not random, this method helps identify significant spatial clusters. Applying this analysis to childhood malnutrition studies can facilitate improved regional targeting by stratifying areas of higher risks and identifying physical locations that are important for the design of nutrition intervention programmes in target regions . TargeteSpatial interpolation is a method of estimating values at an unsampled location using a network of known-valued points . The kriThe selected articles showed that many factors contribute to childhood malnutrition. The determinants of the nutritional condition of children can be classified into several factors, such as: i) proximate factors e.g. age of child, sex of child, birth size, insufficient food intake and infection; ii) distal factors e.g. sociocultural, economic, environmental and climatic variables; and iii) intermediate factors e.g. wealth index, mothers’ ethnicity and level of education of mother and father. Depending on geographical and climatic conditions and anthropogenic influences, these factors are believed to differ temporally and spatially . TherefoThe potential drawbacks of this review are the omission of non-English articles and limited search terms, which could have contributed to the unintended exclusion of studies. This review was dominated by cross-sectional studies. This suggests that the study focused on data collected at a particular point in time, making it difficult to discover patterns in childhood malnutrition status over time. The cross-sectional studies are efficient, inexpensive and simple to conduct; however, they may cause the researcher to be biased in their depiction of the result, which may impact secondary data . A longiMany studies have conducted various spatial analysis methods by combining national and regional childhood malnutrition data and attempts have been made to illustrate the geographic distribution of childhood malnutrition using a map. The methodological strength of such studies spans from mapping and advance statistical spatial analyses through the identification of environmental and other geographical factors on childhood malnutrition. As a result, researchers, policymakers and programme managers have acknowledged location as a crucial determinant of population and health outcomes. This is especially true in the case of childhood malnutrition, which can be impacted by geographical, cultural and environmental factors. According to the findings of this review, the application of spatial analysis methods in childhood malnutrition research is important for understanding the geographic distribution of malnutrition cases, hotspot areas and risk factors associated with childhood malnutrition. To assist the government, policymakers and other authorities in implementing strategies and designing intervention programmes in high-risk areas to reduce malnutrition in children throughout the world, it is necessary to evaluate and understand the geographical distribution of childhood malnutrition cases.


A questionnaire was administered to inquire about demographic, socioeconomic, lifestyle and clinical characteristics. Capillary blood was obtained, anthropometric characteristics were measured and stool samples were collected. As for the secondary objective, anemic adolescents were randomly assigned to an intervention group (nutrition education and iron supplementation) and a control group (iron supplements only). The prevalence of anemia was 37.8%. Female gender, khat chewing, excessive menstruation, and experiencing headaches, fatigue, or dizziness were independent predictors of anemia. In contrast, adolescents who attended private schools, and reported snack consumption or handwashing had a significantly lower risk of anemia. A sample of 116 adolescents participated in the intervention (3 months). Hemoglobin levels were significantly higher in the intervention group compared to the control. Our findings contribute to the identification of high-risk groups that should be targeted by context-specific interventions. The implemented multicomponent intervention may serve as a prototype for larger-scale preventive programs. This study assesses the prevalence and correlates of anemia among adolescents living in the war-affected region of Hodeida in Yemen. A secondary objective was to examine the effect of a nutrition education intervention on hemoglobin levels among anemic adolescents. A cross-sectional study was conducted on a random sample of adolescents aged 15–19 years in Hodeida ( Anemia is acknowledged as a global public health concern, especially in low- and middle-income countries ,2. AnemiAdolescents are recognized as a vulnerable population regarding anemia . This agAt the global level, the prevalence of adolescent anemia is estimated at 15%, but with large disparities between developed and developing countries  . In the Previous studies have reported high prevalence rates of anemia in Yemen, reaching 40% among pregnant women and 49% among children ,29. HoweA cross-sectional study was carried out in 2020 on a random sample of male and female adolescents aged 15–19 years attending secondary schools in the Hodeida Governorate of Yemen. Using a stratified multistage random sampling technique, 5 out of the 26 districts of Hodeida were randomly selected: Al-Hawak, Al-Hali, Al-Mina, Az-Zydiah and Az-Zuhrah. Based on the population size of 16,698 secondary school students enrolled in grades 10, 11, and 12 in these five districts , the estimated sample size for the survey was estimated at 400, with 5% marginal error. The number of 15–19-year-old adolescent students to be selected from each district was determined using the proportional allocation method (based on the number of schools and students in each district). Within the identified districts and schools, subjects were selected randomly from student class lists, based on a systematic random sampling approach. Data collection was initially planned to be conducted in the school setting; however, due to the COVID-19 outbreak, schools were not operational in Yemen . TherefoInclusion criteria were: participants must be aged between 15 and 19 years, and registered at a school in the Hodeida Governorate of Yemen. Adolescents were excluded from the study if they reported having a blood transfusion within 3 months prior to data collection; if they reported suffering from anemia caused by inherited or acquired diseases, or other medical conditions; if they reported to have taken treatments for anemia within 4 weeks prior to data collection; if they reported to be on medication that may interfere with appetite or nutrient absorption. Pregnant female adolescents were also excluded from the study. The study was approved by the Director of Education in Hodeida (25/1/2020). Further approval was obtained from the headmasters of the participating schools prior to data collection. The study was conducted in accordance with the Helsinki declaration, and informed consent was obtained from all research participants and their parents/guardians. A trained researcher conducted one-to-one interviews with the participating adolescents, using a pre-tested multicomponent questionnaire , in addiThe multicomponent questionnaire inquired about the subject’s demographic and socioeconomic characteristics, such as age, gender, type of school , grade, father’s educational level and occupation, mother’s educational level and occupation, monthly family income, and crowding index (ratio of the number of people living in the household over the number of rooms in the household) . The queAnthropometric characteristics including weight (in kilograms) and height (in meters) were measured for all participating adolescents, using a stadiometer and a portable digital scale. Height measurements were obtained without shoes, and body weight was measured to the nearest 0.1 kg with the participant in light indoor clothing and with bare feet or stockings. Anthropometric measurements were interpreted using the World Health Organization (WHO) 2006 criteria  based onPoint-of-care hemoglobin evaluation was conducted using the HemoCue 201 device. One drop of capillary blood was obtained from the participant’s fingertip using a sterile lancet, and placed into a microcuvette, which was later inserted into the electronic hemoglobin device after its calibration to zero. Previous studies have, in fact, validated HemoCue 201 against venous blood measures as the gold standard, and reported high predictability and sensitivity ,34. For Entrobius vermicuaris, Entamoeba histolytica, Giardia lamblia, Ascaris lumbricoides, Schistosoma mansoni, and others.The researcher also provided each participant with a plastic container for the collection of stools. Participants were asked to provide the stool sample after the interview, if possible, after which, the sample was taken immediately to the laboratory for analysis. Other participants provided the sample the second day after the interview. In total, 261 participants provided stool samples for the examination of parasites via the stool ova and parasites test. For this purpose, one drop of normal saline (0.85% NaCl) was placed on the glass slide and mixed with a thin smear of fresh stool. The smear was then examined using a calibrated microscope to detect ova and cysts, using the 20x and 40x objectives. The parasites, and their ova or cysts, were identified according to the shape, size, and internal structure characteristic of the species, which included n = 151) were asked to participate. Those who agreed to participate were randomly assigned into two groups: a control group that received iron supplements (Glovite capsule 200 mg) once daily for 3 months, and the intervention group, which received nutrition education and followed the same iron supplementation protocol as above. In addition, both the control and intervention groups were given two tablets of Mebendazole (deworming medication: Antiver 100 mg) per day for 3 days , prior to the intervention. The educational sessions consisted of four sessions: (1) introducing anemia and the importance of iron, folate and vitamin B12 during adolescence; (2) explaining the risk factors, symptoms, signs, and, consequently, the complications of anemia; (3) providing information about iron-rich foods, enhancers and inhibitors of iron absorption, with a focus on khat chewing and its consequences on iron absorption; and (4) reviewing the previous sessions and providing the students with an educational pamphlet. An intervention trial was conducted to test the impact of a nutrition education program among those who were anemic. Adolescents identified with anemia (A questionnaire exploring anemia-related nutrition knowledge was administered to the participants pre- and post-intervention. The questionnaire inquired about (1) the assessment of anemia; (2) the potential causes of anemia; (3) micronutrient deficiencies that may lead to anemia; (4) signs and symptoms of anemia; (5) foods that are rich in iron; (6) absorption of iron from plant-based vs. animal-based foods; (7) factors that may inhibit iron absorption and factors that may enhance iron absorption; and (8) whether anemia can be prevented and managed via the diet. For each participant, a knowledge score was computed based on the number of correct responses to the knowledge questions, with scores ranging from 0 to 12, the greater the score, the greater the knowledge. Hemoglobin levels were also measured before and after the intervention. During the 3-month period, thirteen anemic adolescents withdrew from this intervention (eight from the control group and five from intervention group), resulting in a study population of 103 adolescents. p-value less than 0.05 indicating that the normality assumption of the data was violated. Anthropometrical Z-scores were calculated by Epi Info software. The sample was divided into anemic vs. non-anemic. Mann–Whitney U tests and Pearson’s chi-square tests were used for comparing differences in the distribution and proportions between anemic adolescents and those who were not anemic, respectively. Covariates were considered for the logistic regression if the p-value was less than 0.05 in the bivariate analysis. Multicollinearity of the covariates was assessed using variance inflation factors (VIFs); a VIF < 10 was considered to show no collinearity between the variables used in the model. Results from the logistic regression analyses are expressed as odds ratios (OR) or adjusted odds ratios (AOR) for anemia with 95% confidence interval (CI). Data were entered and analyzed using Stata . A Shapiro–Wilk test was used to examine the normality of the data distribution of the continuous variables, with a t-tests and one-way ANOVA were used, respectively. A two-tailed p-value of less than 0.05 was considered statistically significant.For the intervention component, particularly for comparing the nutritional knowledge scores and hemoglobin levels at pre- and post-intervention, and between groups, paired As shown in n = 151). Among those with anemia, only one subject  had severe anemia (0.7%), 43% had moderate anemia (7 boys and 58 girls), and 56.3% had mild anemia (45 boys and 40 girls).The mean hemoglobin level (±standard error (SE)) was 12.1 ± 0.1 g/dL, the median (interquartile range (IQR)) was 12.2 (2.1) g/dL, with a minimum of 7.1 g/dL and a maximum of 15.1 g/dL (data not shown). Based on hemoglobin levels, the prevalence of anemia was found to be 37.8% (p = 0.002). Moreover, anemia was more prevalent among those who lived in rural areas (p < 0.001), attended governmental schools (p < 0.001), and whose fathers and mothers completed primary or preparatory school (<0.001). Furthermore, significant differences were noted with regards to monthly family income, whereby 59% of those who belonged to families with low income  were anemic compared to 14.8% among those with an income > YER 70,000 (p < 0.001). p = 0.010). Anemia prevalence was also significantly higher among adolescents who consumed only two meals (62.2%); those who never consumed breakfast (61.8%), snacks (47.1%), or vegetables (61.2%); and those who always consumed fast foods (52.2%) (p < 0.05). Adolescents who reported drinking tea after a period of time post meal were less likely to have anemia (26.5%) compared to those who reported to drink tea immediately after or during a meal (45.9%) (p < 0.001). One-third of adolescents who reported to wash their hands before eating were anemic, compared to 77.5% of adolescents who did not (p < 0.001).p < 0.001). Anemia was also significantly higher among those who had a positive stool test (61.7%) compared to those who had a negative test (38.3%) (p = 0.003). Moreover, anemia was more prevalent among those who experienced excessive menstruation  (p = 0.001) and among underweight  and stunted adolescents .The results of the multivariable logistic regression analysis showed that being female ); khat chewing ); experiencing headaches ), fatigue ), and/or dizziness ); and having excessive menstruation ) were independent predictors of anemia among adolescents . In contn = 58) or intervention group (n = 58). Mean scores of anemia-related knowledge and hemoglobin levels before and after the intervention are shown in For the intervention, the study population included 116 anemic adolescents, randomly sorted in the control (p < 0.001) and hemoglobin levels  post-intervention. Moreover, among those who received the educational intervention, a significant difference was observed in their knowledge scores (76.2 vs. 21.7 over 100) and hemoglobin levels (12.9 g/dL vs. 10.5 g/dL) before and after the intervention.Compared to the control group, those who received the nutrition education intervention had significantly higher knowledge scores  is higher than that reported among 10–19-year-old adolescents in Nepal (31%) , and mor.8% is hiThe fact that, in our study, girls were at a higher risk of anemia, is in line with results presented in the literature . In geneKhat chewing was also found to be an independent predictor of anemia in our study population. Because of its amphetamine-like stimulant effects , khat chThis study also shows that some of the classic symptoms of anemia , includiThe current study shows that adolescents enrolled in private schools had a significantly lower risk of being anemic, and that the prevalence of adolescent anemia in governmental schools 48.2%) was three times higher than in private schools (15.6%). Students enrolled in governmental schools are usually from lower-income households and of lower socioeconomic status , the lat% was thrAscaris lumbricoides infection was lower in children of higher socioeconomic status [In our study, the practice of washing hands was found to be independently associated with lower risk of anemia. Hygiene behaviors, such as handwashing, can in fact reduce the incidence of diarrheal diseases and respiratory infections, which could improve nutritional and micronutrient status . It was c status , and a sc status . This mac status . Some dietary behaviors were found to be associated with the risk of anemia in our study. For instance, and in agreement with previous studies ,75, reguA secondary objective of this study was to test the effectiveness of a nutrition education intervention on anemia-related knowledge and improving hemoglobin levels among anemic adolescents in Hodeida. Prior to the initiation of the intervention, both control and intervention groups were given a course of deworming medication to control intestinal parasitic infestations. In fact, in cases where intestinal parasitic infections are endemic, iron supplementation alone (or in combination with education) will not have the desired impact on anemia reduction ,81 unlesThis study has several strengths. It is, in fact, the first to report on the prevalence of anemia among adolescents in Yemen, a country that is shattered by war, poverty, and humanitarian emergencies. It is also the first to report on sociodemographic, lifestyle, and medical factors that are associated with anemia in this vulnerable population, which can help in the development of future preventive programs. However, the results of this study ought to be interpreted in light of the following limitations. First, the study was restricted to the region of Hodeida, which may limit the generalizability of the data to other areas in the country. Second, the biochemical assessment that was performed allowed for the detection of anemia, without assessing specific nutritional anemias, which limited our ability to report on the prevalence of iron deficiency anemia . In addition, the method of anemia recognition was based on point-of-care hemoglobin estimation using the HemoCue 201 device, instead of complete blood counts. However, it is important to note that several studies have validated HemoCue 201 against gold standards in various populations ,34,84 foThis study is the first to assess the prevalence of anemia and its correlates among adolescents in Yemen, an age group that is recognized as a nutritionally vulnerable population . The stu


Sancao Tiaowei Decoction (SCTWD), a traditional Chinese medicine created by Professor Chen Weijian, has been used in the prevention and treatment of precancerous lesions of gastric carcinoma (PLGC). However, its mechanism has not been made clear. This study aimed to evaluate the therapeutic effect of SCTWD on 1-methyl-3-nitro-1-nitrosoguanidine-induced PLGC in rats and the mechanism of this effect. We found that SCTWD effectively repaired gastric mucosal injury, reversed the process of PLGC, and inhibited the occurrence of gastric cancer to some extent. In the results of hematoxylin-eosin (HE) staining, the number and arrangement of mucosal glands and the number of mononuclear cells in the lamina propria were improved in varying degrees; the enzyme-linked immunosorbent assay (ELISA) showed that the PG I and PGR of the medication treatment group were significantly higher; a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) test showed that SCTWD could significantly upregulate the expression levels of Shh, Ptch, and Gli-1 in the gastric tissue of rats. The immunohistochemical method showed that SCTWD could significantly upregulate the protein expressions of Shh, Gli-1, Smo, cyclin D1, CDKN2A/p16INK4a, and NF-κBP65 and could reduce the expression of Ptch at the same time. Through the preliminary analysis of 75 compounds screened by UPLC-Q-TOF-MS, the main components, such as organic acids, esters and anhydrides, flavonoids, phenols, tanshinones, and so on, have anti-inflammatory and anti-tumor pharmacological effects. The results of KEGG enrichment analysis showed that 5 signaling pathways related to this project were found, and 33 differential genes were presented to construct the interaction network. These results suggested that SCTWD had a good regulatory effect on PLGC and thus may have a multi-targeted effect; SCTWD can not only significantly improve the pathological changes of gastric mucosa in rats with PLGC but also exert a strong effect of the regulation of the hedgehog signaling pathway. Helicobacter pylori, acid inhibition, the protection of the gastric mucosa, and symptomatic treatment (Precancerous lesions of gastric carcinoma (PLGC) are a kind of gastric mucosal histopathological changes that are prone to canceration, including gastric mucosal atrophy, intestinal metaplasia (IM), and dysplasia (Dys). Gastric mucosal atrophy and IM are collectively referred to as chronic atrophic gastritis (CAG) . Accordireatment , but thereatment .Dendrobium officinale Kimura et Migo, inhibits PLGC through suppressing the Harvey rat sarcoma viral oncogene homolog/Phosphatidylinositol 3 Kinase/Protein Kinase B(HRAS/PI3K/AKT) signaling pathway , which has a history spanning thousands of years, has attracted much attention in China and even the whole world. The research of TCM has become a research hotspot in the field of pharmacodynamics and pharmacokinetics. So far, TCM prescriptions have been widely used in the treatment of PLGC disease. For example, it has been reported that Weifuchun (WFC) tablets can regulate the expression of oncogenes and tumor suppressor genes by regulating nuclear factor kappa B (NF-κB), runt-related transcription factor 3/transforming growth factor-β/Smad (RUNX3/TGF-β/Smad), Hh, mitogen-activated protein kinase (MAPK), and Wingless and int-1 (Wnt) signaling pathways , 9. At t pathway . Similary damage . A previy damage . It is wSancao Tiaowei Decoction (SCTWD) is Professor Chen Weijian’s experienced prescription in the treatment of PLGC, which has achieved a good effect in clinical application. In the previous study of this project, the patients with PLGC of the Second Affiliated Hospital of Zhejiang Chinese Medical University who were treated by WC were selected to observe the clinical symptoms and histopathological changes before and after treatment with SCTWD.The results show that SCTWD can not only improve the clinical symptoms of patients with PLGC but also improve the pathological state of the gastric mucosa, repair gastric mucosal injury, and reverse the process of PLGC to some degree in order to reduce the occurrence of gastric cancer. The purpose of this study is to determine the pharmacological effect of SCTWD on PLGC through the PLGC rat model, so as to clarify the therapeutic mechanism of SCTWD on PLGC through the Hh signaling pathway.A total of 36 SD male rats, weighing (125 ± 5) g, were provided by Shanghai Shrek Experimental Animal Co., Ltd., of which animal production license number is: SCXK (Shanghai) 2017-0005; and whose animal use license number is: SYXK (Zhejiang) 2021-0012, raised in the Animal Experimental Research Center of Zhejiang Chinese Medical University, with relatively constant temperature (25°C), constant humidity (75%), and unlimited drinking water and feed.This study has been examined and approved by the Experimental Ethics Committee of The Second Affiliated Hospital of Zhejiang Chinese Medical University and the Animal Experimental Ethics Committee of Zhejiang Chinese Medical University. The design meets the principles of safety and fairness. Experimental animals met the relevant national requirements for medical experimental animals.Chinese Pharmacopoeia. SCTWD is composed of 9 herbs: Pseudostellariae Radix, stir-baked Atractylodis Macrocephalae Rhizoma in bran, Poria, Agrimoniae Herba, Taraxaci Herba, Hedyotis Diffusa Willd, Salviae Miltiorrhizae Radix Etrhizoma, Curcumae Rhizoma, and Glycyrrhizae Radix Et Rhizoma. The herbs were prepared by the laboratory of Zhejiang Chinese Medical University and soaked in water of which the volume was 8 times that of herbs for 1 hour. The traditional Chinese herbs were decocted twice with water whose volume was 20 times, mixed the two-time liquid of the medicine, concentrated into the liquid containing crude drug 2.8 g/ml by a rotary evaporator, which was sterilized and packed separately, then refrigerated for preparation.The traditional Chinese herbs of SCTWD were purchased in the pharmacy of The Second Affiliated Hospital of Zhejiang Chinese Medical University, and the quality was controlled according to the standard of the 2015 edition of 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) was purchased from Hangzhou Northrend Biotechnology Co., Ltd, Hangzhou, Zhejiang, China. Ranitidine was purchased from Hangzhou Kuiteng Biotechnology Co., Ltd, Hangzhou, Zhejiang, China. Xylene (Item No.: R017750-500 ml) was purchased from Shanghai Yien Chemical Technology Co., Ltd, Shanghai, China. Hematoxylin stain Harris (Item No.: BL702A), eosin stain  (Item No.: BL703A), Neutral Balsam (Item No.: BL704A), goat anti-mouse IgG- horseradish peroxidase (HRP) (Item No.: BL001A), goat anti-rabbit IgG-HRP (Item No.: BL003A) were purchased from Biosharp White Shark Biotechnology Co., Ltd, Hefei, Anhui, China. The rabbit kit (enhanced enzyme-labeled goat anti-rabbit IgG polymer) (Item No.: PV-9001), concentrated DAB kit (20 × 2 tubes) (Item No.: ZLI-9017), citrate buffer (Item No.: ZLI9065), and PBS phosphate buffer (Item No.: ZLI-9062) were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd, Beijing, China. The RIPA lysis buffer (Item No.: P0013B), 4-20% SDS-PAGE pre-formed glue (Item No.: P0057A), Western primary antibody secondary antibody removal solution (Item No.: P0025-250ml), actin antibody  (Item No.: AA128), Gli-1 rabbit polyclonal antibody (Item No.: AF6990), Smo rabbit polyclonal antibody (Item No.: AF8010), PTCH1 rabbit polyclonal antibody (Item No.: AF7836), NF-kappa B p65 antibody  (Item No.: AN365), cyclin D1 rabbit polyclonal antibody (Item No.: AF0126), CDKN2A/p16INK4a rabbit polyclonal antibody (Item No.: AF6471), and HRP-labeled goat anti-rabbit IgG (Hendl) (Item No.: A0208) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd, Shanghai, China. PageRuler Prestained Protein Ladder, 10–180 kDa (Item No.: 26617) was purchased from Thermo Fisher Scientific Company, Waltham, Massachusetts, the United States; ammonium hydroxide (Batch No.: 20010328) was purchased from Hangzhou Changzheng Chemical Plant, Hangzhou, Zhejiang, China; sodium chloride (Batch No.: 060304) was purchased from Shanghai No.4 Reagent & H.v.chemical Co., Ltd, Shanghai, China ; 3% H2O2 solution (Batch No.: 200508292) was purchased from Shanghai Broad Peroxide Company, Shanghai, China; and methanol (Batch No.: 200608010) was purchased from Shanghai Zhenxing No.1 Chemical Plant Co., Ltd, Shanghai, China. Anhydrous formic acid (Batch No.: 040518) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd, Shanghai, China; concentrated hydrochloric acid (Batch No.: 20050603) and ethanol (Batch No.: 20050521) were purchased from Hangzhou Chemical Reagent Co., Ltd, Hangzhou, Zhejiang, China; paraformaldehyde (Batch No.: 20051208) was purchased from Tianjin Institute of Chemical Testing Agents Quzhou Juhua Co., Ltd, Tianjin, China; the neutral formaldehyde buffer solution (Batch No.: 20060101) was purchased from Quzhou Juhua Co., Ltd, Quzhou, Zhejiang, China. The rabbit anti-patched antibody (Item No.: bs-1614R) is purchased from Beijing Boosen Biotechnology Co., Ltd, Beijing, China; the Shh antibody (item No.: 20697-1-AP) was purchased from Proteintech Group Co., Ltd, Chicago, Illinois State, the United States. The rat serum TNF-α enzyme-linked immunosorbent assay (ELISA) kit (Item No.: BEP30635), rat serum pepsinogen I (PG I) ELISA kit (Item No.: BEP30821), rat serum pepsinogen II (PG II) ELISA kit (Item No.: BEP30896), and rat serum gastrin (GAS) ELISA kit (Item No.: BEP30675) were purchased from Shanghai Langton Biotechnology Co., Ltd, Shanghai, China. The rat IL-6 kit (Item No.: AB234570) was purchased from Abcam Company Co., Ltd, Cambridge, the United Kingdom. The rat IL-8 ELISA kit (Item No.: SEKR-0071) was purchased from Shanghai Solebo Technology Co., Ltd, Shanghai, China. While IL-8 Antibody-Internal (Item No.: DF6698) and IL-6 Antibody-Internal (Item No.: DF6087) were purchased from Affinity Biosciences Co., Ltd, Cincinnati, State of Ohio, the United States. The RNA extraction kit (Item No.: AHF1991D), RT reverse transcription kit (Item No.: RR036A), SYBR Green fluorescent quantitative PCR dye (Item No.: AK9301) were purchased from TaKaRa Bio Co., Ltd, Tokyo, Japan.Pseudostellariae Radix 15 g, stir-baked Atractylodis Macrocephalae Rhizoma in bran 15 g, Poria 15 g, Agrimoniae Herba 15 g, Taraxaci Herba 15 g, Hedyotis Diffusa Willd 15 g, Salviae Miltiorrhizae Radix Etrhizoma 15 g, Curcumae Rhizoma 9 g, and Glycyrrhizae Radix Et Rhizoma 9 g were immersed for 30 min in 10 times volume of pure water, decocted and extracted twice for 1 hour each time, and the filtrate was concentrated to obtain the extract with the mass concentration of 1.23 g/ml. Approximately 1.0 ml of the extract was taken, which was put into the 100 ml measuring bottle, and deionized water was added for 30-minute ultrasonic. Deionized water was added to fix the volume until the concentrated solution is completely dissolved, it was centrifuged for 20 min with 14,000 r/min, and the supernatant was filtered through a 0.2 μm microporous membrane. All samples were analyzed by UPLC-Q-TOF/MS high-resolution four-stage flight tandem liquid chromatography–mass spectrometry (American model Waters SYNAPTG2-Si) for positive and negative ion mode analysis.Chromatographic conditions: Eclipse Plus C18 column  was selected as the chromatographic column. The separated mobile phase consists of solvent A  and solvent B . The gradient elution conditions are as follows: gradient elution is used , and the later running time is 5 min. The injection volume is 2 μl, the temperature of the column incubator is 35°C, and the temperature of the automatic injector is 10°C.Mass spectrometry conditions: The ESI positive ion mode and negative ion mode were used to collect data. The range of scanning (m/z) was 50–1,200 Da, the capillary voltage of positive ion mode was 3,000 V as well as the negative ion mode was 2,500 V, the source offset voltage was 80 V, with the taper hole voltage 40 V and the atomizer pressure 6.0 Bar, the source temperature was 100°C, the desolvent temperature was 400°C, and the desolvent gas flow rate was 800 L/h.Experimental instrument: UPLC-Q-TOF-MS  was purchased from Waters Corporation, Milford, Massachusetts, United States.Thirty-six 3-day-old male suckling rats were selected and divided into cages by the random number table method after routine feeding to 3-week-old weaning. A total of 8 suckling rats were divided into the blank group and free diet. The other 28 suckling rats were used as model rats, and the model group was fed with nitrosoguanidine carcinogen combined with ranitidine feed. Approximately 420 ml of pure water mixed with 80 ml of alcohol and 5 g of nitrosoguanidine formed the mother liquid of 10 mg/ml, and the drinking water containing 100 μg/ml of medicine was prepared according to 1:100 once a week, combined with 0.03% ranitidine feed. The model group was fed, following the rule “full for two days and fasting for one day”. The model was made continuously by the above-mentioned method for 10 weeks. In the model group, 2 rats were randomly selected for anesthesia, and the stomach was taken for pathological examination every 2 weeks. After the establishment of the model, the model rats were randomly divided into two groups: the model group (n = 9) and treatment of SCTWD group (n = 9). The pathology was reviewed and checked by senior pathologists to determine gastric mucosal atrophy, and IM and IN were seen at the same time; then, the PLGC model was judged to be successful.Referring to 60 kg, the average body mass of adults, according to the dose conversion between the clinical drug dose and the animal dose, the dose given SCTWD daily with raw medicine was 22.4 g/kg (8 ml/kg). The model group was given physiological saline, according to the body weight of rats. The intragastric treatment of 0.1 ml/(kg·d) was performed. Drug intervention lasted for 16 weeks.The rats were weighed and recorded every other day before the establishment of the model and during the experiment, and the changes of weight were observed.After giving the last medicine, the rats were treated with fasting for solids and liquids for 12 h and then anaesthetized with 3% pentobarbital sodium; 10 ml of blood was collected from the celiac vein and stored at 4°C. The 5 ml separated serum was packed in 3 parts, stored in the refrigerator at -80°C. The gastric tissue was cut along the great curvature of the stomach with the gastric contents removed, and spread out for general observation. The gastric tissue at the boundary of the lesser curved sinus was taken for general observation, with one part fixed with 4% paraformaldehyde, as well as the other three parts were preserved at -8°C, and the alcohol was dehydrated step by step, transparented by xylene, embedded by paraffin, and made routinely into paraffin sections to be used.After the gastric body was embedded in paraffin, the gastric body was sliced with slicer and stained with HE, and the histopathology of gastric mucosa of rats in each group was observed under light microscope.After grinding, RNA was extracted with Trizol reagent and trichloromethane, precipitated with isopropanol, washed with 75% ethanol, dried and dissolved in a proper volume (30~50 μl) of diethyl carbonate containing coke (DEPC), and stored at -80°C. Total RNA was extracted directly for PCR amplification. According to the instructions of the M-MuLV first-strand cDNA synthesis kit, the reverse transcription was complement deoxyribonucleic acid (cDNA). After the reverse transcription was completed and using this as a template, THUNDERBIRD SYBR qPCR Mix and specific primers were used to synthesize the corresponding PCR products. The specific operation steps refer to the instructions of the kit. Heat the product for 10 min at 70°C to end the reaction, and put it on ice for follow-up experiments or cryopreservation. The RT-PCR reaction conditions were pre-denatured at 94°C, 5 min; at 94°C, 50 s; at 58°C, 50 s; at 72°C, 1 min. Approximately 8 min was extended at 72°C after 35 cycles. The Ptch reaction procedure was pre-denatured at 94°C, 5 min: at 94°C, 50 s; at 54°C, 50 s; at 72°C, 1 min. Approximately 8 min was extended at 72°C after 35 cycles.Experimental instrument:1300 Series A2 Class II, Type A2 Biological Safety Cabinets, High Speed Refrigerated Centrifuge, Ultra-Low Freezers and Nanodrop Trace Nucleic Acid Tester were purchased from American Thermo Fisher Scientific company, Waltham, Massachusetts, the United States. Homogenizer was purchased from Bertin Technologies, Aix-en-Provence, France. Automatic Fluorescence Quantitative PCR Instrument was purchased from American ABI company, Waltham, Massachusetts, the United States. was purchased from Bio-Rad Laboratories, Hercules, California, the United States. SIM-F140AY65-PC Ice Maker was purchased from Panasonic company, Osaka, Japan. Pipette was purchased from Eppendorf company, Hamburg, Germany. UVITEC Ultraviolet Gel Imaging Systemwas purchased from British UVITEC Company, Cambridge, the United Kingdom.The levels of PG I and PG II in the blood of rats were detected by double-antibody-sandwich ELISA, and the ratio of PGR was calculated. The specific operation was carried out according to the instructions of the kit.Experimental instrument: Multiskan FC Microplate Photometer was purchased from American Thermo Fisher Scientific company, Waltham, Massachusetts, the United States. Plate washer was purchased from Bio-Rad Laboratories, Hercules, California, the United States. Liquid Transfer Machine was purchased from Eppendorf Company, Hamburg, Germany.After paraffin section, using xylene for dewaxing to water, gradient rehydration and high pressure antigen repair, immunohistochemical staining was performed according to the instructions of the kit. PBST buffer staining was used as negative control. Five images of each tissue about mucous layer were randomly collected under a ×200 microscope, and the images were analyzed by Image Pro Plus 6.0 software.Experimental instrument: Nikon Eclipse 80i Microscope nd DS-H1 500 Pixel CCD were purchased from Nikon Corporation, Tokyo, Japan. Carl Zeiss lmaging Systems was purchased from Carl Zeiss Company, Oberkochen, Germany. Microwave oven was purchased from Guangdong Galanz Enterprises Co., Ltd, Foshan, Guangdong, China. DRP-9052 type electric thermostat was purchased from Shanghai Senxin Experimental Instrument Co., Ltd, Shanghai, China. BG-270 waterproof electric thermostat was purchased from Medical Equipment Factory of Shanghai Medical Instruments Co., Ltd, Shanghai, China.2 FC ≥ 1) was used as the change threshold, and P < 0.05 was used as the criterion for screening differential genes. In the set comparison group, the results of differentially expressed genes were obtained, and differential genes were screened to construct interaction networks.In this project, the difference multiple FC ≥ 2 or FC ≤ 0.5  was statistically significant. The UPLC-Q-TOF-MS method was analyzed by Waters Mass Lynx 4.1 and MSE Data Viewer 1.2 software.All the data were analyzed by SPSS 26.0 , and expressed by mean ± standard error (SEM). The measurement data were evaluated by ANOVA analysis of variance, and pairwise comparison was conducted by the Dunnet test. The values of the statistical results were retained in the last 2 decimal places. For data analysis and processing, each group is allowed to eliminate no more than 2 abnormal data . The difference . After 6 months of treatment with SCTWD, the clinical symptoms and histopathological changes of the patients were significantly improved. The results of clinical symptom improvement are shown in P < 0.01); there was significant difference in symptom before and after treatment, such as insomnia, gastric upset, sticky and greasy in mouth, body trapped, and hypochondriac pain (P < 0.05); the lack of warmth in the limbs was no significant difference between before and after treatment (P > 0.05). The total effective rate of symptom improvement is 91.96%; the total effective rate of atrophy is 62.89%; the total effective rate of intestinal metaplasia is 74.31%; and the total effective rate of dysplasia is 61.54%. The gastroscope and pathology of the patients before and after treatment are shown in In the previous study of this project, 112 patients with PLGC were selected from The Second Affiliated Hospital of Zhejiang Chinese Medical University, which is the outpatient clinic of Professor Chen Weijian, from January 2019 to June 2020, as shown in The difference of weight of rats weight difference of rats at the beginning of treatment and the 12th week and 16th week of treatment were calculated, respectively, and the difference of weight is shown in The glandular stomach of rats in the blank group had no obvious change; the mucous membrane was covered with more mucus and the mucosa was ruddy, while in the model group, the glandular stomach had some changes, the mucosal surface was yellow, and the color of exposed mucosa was slightly whiter than that in the blank group.The results are shown in P < 0.01), and there was no significant difference between the treatment group, the model group and blank group , and there was no significant difference between the treatment group and the blank group , and there was no significant difference between the blank group and the treatment group . Meanwhile, compared with the blank group, the expressions of Shh protein in the gastric tissue of the model group were increased, but the gene expressions of the medication treatment group were lower than that of the model group (P < 0.05). On the contrary, the expression of Ptch was significantly decreased in the model group and upregulated in the medication treatment group (P < 0.05).The results are shown in P < 0.01, The positive expression of Smo was mainly located on the cell membrane and in the cytoplasm. Microscopically, positive cells were expressed in the mucous layer, mucosal muscular layer, muscular layer, and serous layer of gastric tissue. Compared with the blank group, the expression of Smo, interleukin-6 (IL-6) and interleukin-8 (IL-8) protein in the gastric tissue of the model group increased, but compared with the model group, the gene expression of the medication treatment group decreased significantly(2 FC ≥ 1) as the threshold and P < 0.05 was used as the criterion for screening differential genes to count the number of differential genes in all comparison groups. Comparing the model group to the blank group, the results showed that 343 genes were downregulated and 215 genes were upregulated. A total of 600 genes were downregulated and 436 genes were upregulated upon comparing the medication treatment group with the model group, which was shown in a bar chart  classification map, five signaling pathways related to this project are shown in the In the literature of TCM, there is no clear discussion on PLGC, which can be classified into the categories of “ruffian fullness,” “epigastric pain,” “stomach ruffian,” and “gastric discomfort” according to its clinical manifestations. Professor Chen Weijian believes that the pathogenic factors of PLGC are complex and diverse, such as the invasion of exogenous pathogen, the internal injury of diet, emotional disorder, and the weakness of spleen and stomach, which can cause the ascension and descension of spleen and stomach and the dysfunction of the qi of the middle energizer. The disease is located in the stomach and is closely related to the spleen and liver. The disease is mostly based on the deficiency in origin and excess in superficiality, intermingled deficiency and excess: the deficiency of spleen and stomach, spleen qi deficiency and stomach yin deficiency, and the internal resistance of exogenous pathogen, such as blood stasis, qi stagnation, and dampness-heat. Its basic pathogenesis is the weakness of spleen and stomach, the stagnation of qi, blood, and phlegm, which obstructs stomach collaterals, leading to the loss of the nourishment of the stomach body and the onset of the disease. The pathological manifestations were abnormal metabolism of gastric mucosal cells, the atrophy of the gastric gland, IM, and even low-grade intraepithelial neoplasia, which eventually developed to gastric malignant tumor.H. pylori infection and “improper diet” are the main causes of “inflammatory-cancer transformation” , so it can be taken for a long time without hindrance. Hedyotis Diffusa Willd has a taste of bitter cold, which has the effect of clearing heat, detoxification, and dampness. The taste of Agrimoniae Herba Ledeb is bitter and astringent, and it has the function of astringent hemostasis, detoxification, and tonifying deficiency. Poria enters the heart, lung, spleen, and kidney meridians, which are sweet and can tonify, lighten, and permeate turbid toxin. All three herbs mentioned above are ministerial drugs, which are combined with Pseudostellariae Radix and stir-baked Atractylodis Macrocephalae Rhizomae in bran to tonify without stagnation, to clear heat, and to detoxify to remove dampness. Experience Gained in Treating External Diseases shows that “Tumor is formed by blood stasis, turbid qi and phlegm stagnation of five internal organs. Curcumae Rhizoma has the function of breaking blood, removing blood stasis and relievig pain, and Salviae Miltiorrhizae Radix Etrhizoma has the function of promoting blood circulation, removing blood stasis, and relieving menstruation and pain, which are adjuvant drugs. Glycyrrhizae Radix Et Rhizoma, as an adjuvant and messenger drug, is to tonify spleen and middle qi, help Pseudostellariae Radix and stir-baked Atractylodis Macrocephalae Rhizoma in bran nourishing middle warmer and tonifying qi, and even become more adjustable and compatible with all kinds of drugs. The combination of various medicines can play the role of eliminating and tonifying which shows the effects of strengthening the spleen and stomach, regulating qi, resolving phlegm, removing blood stasis and promoting tissue regeneration.” Modern pharmacological studies have shown that Agrimoniae Herba, Taraxaci Herba, Hedyotis Diffusa Willd, Salviae Miltiorrhizae Radix Etrhizoma and Curcumae Rhizoma all have the effects of treating gastrointestinal diseases and anti-cancer. Taraxaci Herba extract has a strong ability to activate delayed anaphylaxis of tumor cells and can inhibit the invasion and migration of gastric cancer cells , 22.4 g/kg (8 ml/kg), and 44.8 g/kg (16 ml/kg) with raw medicine. All the three concentrations included the range of safety. Based on the results of ELISA experiment, we chose the middle-dose group for other experiments because its effect is the best out of the three groups.In the occurrence of PLGC, the existence of the Hh signaling pathway  is very This study found that the Hh signaling pathway is activated in the occurrence and development of PLGC. After the intervention of SCTWD, the expression levels of key factors that are Shh, Smo, Gli-1, and so on in the signaling pathway are downregulated, while the expression of Ptch is upregulated, indicating that SCTWD can regulate the activation of the Hh signaling pathway by regulating the protein expression levels of multiple target molecules in the Hh signaling pathway, improve the pathological changes of gastric mucosa, and inhibit the progression of PLGC in PLGC rats.Currently, it’s generally believed that PLGC is an irreversible and refractory disease because of the lack of specific and effective drugs in clinical practice. The current treatment methods can only delay the process, which means that there is no effective standard treatment strategy. TCM is more effective than western medicine in improving clinical symptoms, repairing mucosal injury, and reversing PLGC gastric gland atrophy and IM. In recent years, more and more scholars have found that the Hh signaling pathway plays an important role in cell proliferation, cell differentiation, and organ development during embryonic development, and its abnormal activation is related to a variety of human cancers. The multi-target effect of TCM is the main development direction of TCM in the treatment of PLGC to prevent the occurrence of gastric cancer. It has become a new trend to clarify the pathogenesis of PLGC and the effect mechanism of prescription and medicine.This study points out that spleen and stomach weakness and qi–blood–phlegm–dampness-blocking stomach collaterals are the basic pathogenesis of PLGC. Taking “invigorating the spleen and stomach, regulating qi, resolving phlegm, removing blood stasis and regenerating” as the treatment principle, the concept of strengthening the body resistance to eliminate pathogenic factors through its treatment, while regulating the expressions of proteins related to the Hh signaling pathway, is the main basis of the prescription of SCTWD for the differentiation and treatment of PLGC. In conclusion, SCTWD has a significant regulatory effect on MNNG-induced PLGC and may have a multi-targeted effect. SCTWD can not only significantly improve the pathological changes of gastric mucosa in rats with PLGC but also exert a strong effect of the regulation of the Hh signaling pathway.https://figshare.com/, 10.6084/m9.figshare.17311556.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 studies involving human participants were reviewed and approved by the Experimental Ethics Committee of The Second Affiliated Hospital of Zhejiang Chinese Medical University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Animal Committee of the Animal Experimental Center of Zhejiang Chinese Medical University.YaC, YiC, SC, LZ, and QS conceived and designed the study. YaC, YiC, YZ, and TY performed the experiments. YaC and YiC analyzed the data. WC and BC contributed reagents and materials. YaC and YiC wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by the Traditional Chinese Medicine Modernization Special Project of Zhejiang Province (Project No.: 2022ZX007).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.


To explore the patterns of the exposure-response relationship between arsenic exposure and cardiovascular disease (CVD) mortality and investigate the effect of cigarette smoking on the association.Seven thousand seven hundred thirty-five tin miners with at least 10 years of arsenic exposure were enrolled since 1992 and followed up for 27 years. Each individual's air arsenic exposure at workplace was calculated by time weighted average arsenic concentration × exposure months. Detailed information on smoking was collected at baseline, and information on smoking status was collected for five consecutive years from 1992 to 1996. Hazard ratio (HR) and 95% confidence interval (CI) for the risk of CVD were estimated using Cox proportional hazards models.3), 1.29 (95%CI: 1.02–1.67) in 99.5–361.0 mg/m3. Further, participants with age at first exposure <18 years had a significantly higher risk of morality from CVD, cerebrovascular and heart diseases than those with ≥18 years. Finally, all synergy indices were greater than 1 , indicating that the joint effect of arsenic exposure and cigarette smoking on CVD mortality was greater than the sum of their individual effect.A total of 1,046 CVD deaths occurred in this cohort over 142,287.7 person-years of follow up. We firstly reported that for equal cumulative exposure, participants exposed to higher concentrations over shorter duration had a higher risk of CVD mortality than those exposed to lower concentration over longer duration. The HR and 95% CI were 1.38 (95%CI: 1.03–1.85) in participants exposed to arsenic concentration (45.5–99.5 mg/mExposure to air arsenic at workplace is adversely associated with mortality from CVD, especially among smokers younger than 18 years and smokers. Environmental exposure to high concentration arsenic had adverse health effects including cancers , cardiovIn arsenic-related CVD studies, smoking information should be taken into consideration. This is because cigarette smoking is also a risk factor for CVD and accounts for 10% of CVD-related deaths worldwide . AlthougIn 1973, the nationwide survey on the cause-of-death in China revealed that Gejiu, a city located in Yunnan Province in southern China, had the highest male lung cancer mortality rate in all of China. The disease burden was involved principally in the tin miners around the city of Gejiu. Since 1986 the Nation Cancer Institute/National Institute of Health had collaborated with scientists at Cancer Hospital, Chinese Academy of Medical Sciences and the Labor Protection Institute to conduct a number of case-control and cohort studies among the tin miners, they found that the phenomenon that these miners lacked effective protective equipment and the factory lacked necessary ventilating facilities, which made miners exposed to extremely high concentrations of environmental radon and arsenic pollutants (accompanied by the mining process of tin ore) –13. In 1With an extend follow-up, we firstly explore the patterns of the exposure-response relationship between arsenic exposure and CVD mortality in this prospective cohort study; And, with information on smoking at multiple points in time, we also investigated the synergy between arsenic exposure and cigarette smoking on CVD mortality.This cohort is a combined cohort. Briefly, a total of 3,278 tin miners with at least 10 years of arsenic exposure were enrolled into the arsenic occupational cohort, and 6,017 tin miners with at least 10 years of underground radon exposure were included into the radon occupational cohort. Given tin miners exposed to both radon and arsenic simultaneously, we also collected air arsenic exposure data in the radon cohort. Therefore, we combined arsenic cohort and radon cohort together into the full cohort . Then, 161 former smokers were excluded, due to instability of smoking status. One thousand three hundred ninety-nine tin miners with a diagnosis of CVD or diabetes at baseline were also excluded. Finally, 7,735 tin miners were included into the final analysis .A detailed occupational history included information on job title, worksite, and starting/stopping dates by year for eachjob held at the Yunnan Tin Corporation (YTC) for at least 1 year.Since 1986, we have conducted lots of researches about the arsenic-related health effects in these tin miners. To measure the cumulative arsenic exposure of individual miners, we collected arsenic exposure concentration in the working environment of different workplaces in different years, then investigated the occupational history of miners including the workplaces and working hours. Finally, we multiply the above two factors to obtain individual arsenic exposure data. Therefore, the descriptions of arsenic exposure in all our studies are the same and detailed information had been reported previously . Briefly3, then fell appreciably to about 6.2 mg/m3 around 1964 when the practice of wet drilling became widespread. Typically, arsenic represents about 1.34% of the mined ore by weight as trivalent arsenic (As2O3).Airborne dust concentrations at the YTC were first measured in the 1950s, when dry drilling was common and large scale mining was taking place. Underground airborne dust concentrations reached 20–102.6 mg/mAirborne arsenic concentration before 1970 was estimated as the product of arsenic content of dust times the concentration of dust in the air. Airborne time weighted average arsenic concentration was calculated for ambient air in the mining environment for each mine and smelter based on the arsenic content of dust, the concentration of dust in the air, and the specific time. After 1970, direct air arsenic concentrations were measured. Calculated or measured mean values by era are shown in 3) × exposure months. One IAEM is equivalent to exposure for 25 days (one month) at 1 mg/m3. If total inspired air per day is estimated at 3.6 m3 and 100% absorption is assumed then one IAEM is equivalent to 90 mg total arsenic exposure.Individual worker exposure to arsenic for each job was estimated by using an index (index of arsenic exposure month or IAEM) as follows: IAEM = time weighted average arsenic concentration  for radon daughters as WL (working level) for the three eras are summarized in Mine-, job-, and era-specific exposure to radon daughters was estimated as WLM for each job for each subject using the following formula:Cumulative exposure was estimated by summing the exposure levels from each job before diagnosis for cases or to the matched case for controls.Definition of cessation of arsenic exposure: For retired miners, the date of cessation of exposure was the date of retirement; for unretired workers, the date of cessation was set to 1996. This date was selected because decades of engineering protection measures dramatically reduced arsenic exposure levels similar to the Chinese national hygiene standard levels by 1996 .Effect modification factors: We defined time since last exposure (TSE), attained age (AA), and age at first exposure (AFE) as effect modification factors of arsenic-related CVD.All miners were enrolled at baseline (in 1992) and a self-designed and standardized questionnaire was administrated to each participant to collect information on smoking through a face-to-face interview with well-trained local health workers. A total of 13 questions included information on smoking status, what types of tobacco products they used , the age they started/stopped smoking, number of cigarettes/waterpipe/long-stem pipe smoked per day. According to the smoking status at baseline, we divided miners into smokers, former smokers, and never smokers. At baseline, individuals who had smoked regularly for 6 months or longer at any time in their lives were classified as smokers, and those who have a smoking duration of less than 6 months were considered never smokers and smokers who ceased smoking at enrollment were former smokers.In addition, to test the stability of smoking status, a self-designed questionnaire was administrated to each miner to collect information on smoking status and type of tobacco for five consecutive years from 1992 to 1996. The change of smoking status for at least two consecutive years was identified as “the real behavior change” from 1992 to 1996.Then, results showed that only 110 of 6,899 (1.6%) of current smokers quit, and only 31 of 1,463 (2.1%) of never smokers started smoking. However, 161 of 933 (17.3%) former smokers returned to smoking.The impact of type of cigarette was not considered in final analysis. In addition, we calculated a cigarette-equivalent variable that combined cigarette and water-pipe use by adjusting moderately that 1 g water-pipe = 1 g cigarette , 14, 19.The primary outcome was deaths from CVD, defined as ICD-10  I05-I69 in cohort participants from baseline to December 31, 2018. The secondary outcome was deaths from cerebrovascular diseases (ICD-10: I60–I69) and heart diseases . Other causes of death were viewed as a competing event.Causes of deaths were identified from the local cancer registration agency, medical record system, death cause systems of public security bureau, and funeral parlor, and face-to-face interviews with relatives and workmates of the participants. In the process of information extracting, participants' name, age, work units and home address were taken into consideration. By the end of December 31, 2018, 136 participants (1.8%) were lost to follow-up, with a follow-up rate of 98.2%. Total causes of death and data sources in this cohort was seen in 3 (Group 1), 1,876 in 2.5–4.4 mg/m3 (Group 2), 2,032 in 4.5–13.9 mg/m3 (Group 3), 1,924 in ≥14.0 mg/m3 (Group 4).We tabulated baseline frequencies and percentages by demographic factors for participants in the different arsenic concentration groups, which all miners were equally classified as four subgroups by arsenic concentration: 1,903 participants in <2.5 mg/mPerson-years was computed from the date of enrollment to the date of death from any cause or December 31, 2018, whichever came first. In the Model 1, we modeled the association between cumulative arsenic exposure/arsenic concentration and risk of CVD mortality, and Cox proportional hazard models were used to calculate hazard ratio (HR) and 95% confidence intervals (CI) for each factor, adjusting for sex and baseline age groups (5-years), radon, BMI, smoking information , educational level . The lowest exposure (Group 1) subgroup was selected as the reference group during statistical analysis. In addition, to explore patterns of the exposure-response relationship of arsenic-related CVD, we also compared the risks of a higher concentration at shorter duration with a lower concentration at longer duration within a fixed cumulative exposure. Finally, we modeled the modification effects of TSE, AA and AFE on arsenic-related CVD mortality. The median of 25.5 years for TSE and 50 years for AA were selected as the cut-off points to ensure sufficient sample size for statistical analysis, and the first exposure of 18 years old as the cut-off value to define the initial exposure in childhood (<18 years) or adulthood (≥18 years). And the subgroups  were selected as the reference group.3, 2,696 in 3.0–8.0 mg/m3 and 2,584 in 8.1–100.2 mg/m3. In addition, current smokers were divided into two subgroups by the median of 25 pack-years. All analysis was conducted with SAS 9.4 .The joint effect of arsenic and cigarette smoking was estimated by the synergy index , which wAs shown in We found an increased risk of CVD mortality with increasing levels of either cumulative arsenic exposure or concentration. The same trends were observed in mortality from cerebrovsacular and heart diseases.3. For heart diseases, the HR and 95% CI were 1.59  in participants exposed to 99.5–160.9 mg/m3; However, the confidence intervals are broadly over lapping and the data are not significantly different in other exposed subgroups, thus the diseases  mortality risks are similarly consistent for participants exposed to higher concentrations over shorter duration as compared with lower concentrations over longer duration.For a fixed level of cumulative arsenic exposure, participants exposed to higher concentrations of arsenic over shorter duration had a higher risk of CVD mortality than those exposed to lower concentration over longer duration in participants exposed to 45.5–99.5 and 99.5–361.0 mg/mIn addition, for a fixed level of arsenic concentration, any significant associations were not shown between cumulative arsenic exposure and the risk of deaths from any CVD, cerebrovascular or heart diseases  as modifiers of the arsenic exposure–response relationship. Within a fixed arsenic concentration, participants with age at first exposure lower than 18 years had a significantly higher risk of mortality from CVD, cerebrovascular and heart diseases than those with 18 years or above. Similarly, the significant variations were seen in the effect modification of time since last exposure, and those with lower than 25.5 years had a higher mortality risk than those with 25.5 years or above. However, for attained age, although the HR > 1 in most exposed subgroups, there was no statistically significant variations when compared participants lower than 50 years and those older than 50 years or above. These results were shown in 3) had an increased risk of cardiovascular death , cerebrovascular death  or heart death , when compared with those with the lowest level (0.1–2.9 mg/m3). Among participants with the lowest arsenic level, those who had 25 or more pack-years of smoking had an increased risk of cardiovascular death , cerebrovascular death  or heart death , compared with nonsmokers. When compared with nonsmokers with an arsenic exposure level among 0.1–2.9 mg/m3, those who with an arsenic level of 8.1–103.2 mg/m3 and smoked for more than 25 pack-years had an increased risk of cardiovascular death , cerebrovascular death  or heart death . Among participants with the highest arsenic level, HR >1 for all three outcomes regardless of smoking status.As shown in In addition, 15%−43% of cardiovascular death, 1%−68% of cerebrovascular death and 20%−74% of heart death were attributable to both arsenic exposure and cigarette smoking. Furthermore, all synergy indices were >1  indicating the existence of synergism in an additive way .Based on this occupational cohort with a long-term follow-up time, we explored the patterns of the exposure-response relationship between airborne arsenic exposure and CVD mortality and investigated the synergy effect of cigarette smoking. Data showed that participants with <18 years at first exposure, <25.5 years since last exposure and smoking were more prone to deleterious effect of airborne arsenic exposure.Consistent with other previous studies –26, we fThis cohort firstly reported that the arsenic-related risk of deaths from CVD from initial exposure in childhood (<18 years) was greater than when first exposure occurred at adulthood (≥18 years), which further strengthened evidence from a cohort study in Matlab, Bangladesh with 13 years follow-up and <40 CVD death cases . In the In addition, our data suggested that arsenic-related risk of deaths from CVD, cerebrovascular or heart diseases declined with time since last arsenic exposure. For the long-term health impacts of arsenic exposure, an ecological study in Northern Chile showed that arsenic exposure had very long latency, because the risks of lung, bladder, and kidney cancers still increased at least 40 years after exposure reduction . In our 3, and they would have a higher risk of CVD deaths compared with reference group.Our data suggested that cigarette smoking increased susceptibility to the CVD effects of arsenic, which was similar to findings from the Health Effects of Arsenic Longitudinal Study in Bangladesh  and the In addition, the synergy between smoking and arsenic exposure on CVD deaths persists either in light smokers or heavy smokers. Moreover, for equal arsenic concentration, variations in CVD deaths are not statistically significant when compared light smokers with heavy smokers. More and more evidences support that smoking cessation, but not reduction, is associated with reduced CVD risk –46. TherA major strength of this study was that a clear-cut occupational arsenic exposure pattern made it possible to evaluate the relationship between lifetime exposure and CVD deaths. For retired miners in our cohort, occupational arsenic exposure was negligible because miners left the workplace which was the main resource of pollutant. In addition, we tracked changes in smoking status during the initial four years of follow-up, which strengthened the stability of smoking information. Lastly, this cohort over 20 years of follow-up with little loss to follow-up had relatively large sample size, which resulted in sufficient statistical power in subgroup analysis. This study also had several limitations. Inclusion criteria with 10+ years of arsenic exposure history meant that exposed miners were be served as a reference category, which would underestimate our estimates of the arsenic effect. Secondly, in our cohort, the high smoking rate (76.9%) resulted in significant passive smoking exposure, even for non-smokers. Therefore, the use of “non-smokers” with such exposure would also bias estimates toward the null. Finally, individual miners' arsenic exposure was obtained through the individual exposure time and airborne dust concentrations in workplace, but there was no validation or quality control of different collection methodologies at different times. Therefore, the uncertainty of this estimation might bias our results, and urine arsenic concentrations should be measured in the future studies.In summary, for equal cumulative arsenic exposure, more attention should be paid to protect population with higher concentration over shorter duration from CVD mortality. And, more attention should also be given to those with <18 years at first exposure, <25.5 years since last exposure and tobacco users.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was approved by the institutional review boards of the National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences (201812190401002). The patients/participants provided their written informed consent to participate in this study.Y-LQ and F-HZ had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Conception and design: Q-HZ and Y-LQ. Data collection: ZS, Y-GF, and Q-HZ. Analysis, interpretation, and drafting the article: ZS and X-HJ. Manuscript revision: Y-GF, Q-HZ, and Y-LQ. All authors contributed to the article and approved the submitted version.