The Tokyo 2021 Olympic Games was anticipated to expose athletes to the most challenging climatic conditions experienced in the history of the modern Olympic Games. This study documents strategies executed by Australian endurance athletes during the team holding camp and Olympic Games experiences, including (1) baseline physiological data, training data, and heat acclimation/acclimatization practices; (2) pre- and in-race cooling and nutritional strategies, and (3) Olympic Games race performance data.2max 63.2 ± 8.7 mL⋅kg–1⋅min–1; sum of 7 skinfolds 53.1 ± 23.4 mm) were observed prior to and during the team holding camp held in Cairns, QLD, Australia. Athletes completed 6–7 weeks of intermittent heat acclimation training, utilizing a combination of 2–4 passive and active acclimation sessions per week. Active acclimation was systematically increased via exposure time, exercise intensity, temperature, and humidity. In the team holding camp, athletes undertook a further 23 heat acclimatization training sessions over 18 days in a continuous fashion. Hyperhydration (using sodium and glycerol osmolytes), and internal and external pre-and in-race cooling methods were also utilized. A low energy availability intervention was implemented with two athletes, as a strategy to periodize ideal race body composition. Race performance data and environmental conditions from the 2021 Olympic Games were also documented.Six athletes , which was within 2% of her 20 km personal best time, and 8th place in the men’s 50 km event (3:52:01), which was a personal best performance time.The highest values for aerobic capacity were 63.6 mL⋅kgPeriodized training, heat acclimation/acclimatization, cooling and nutritional strategies study may have contributed to the race outcomes in Olympic Games held hot, humid conditions, for the race walkers within this observational study. It was projected that the Tokyo Olympic Games would expose athletes to the most challenging climatic conditions ever observed in the history of the modern Olympic Games . In respPrior to major international championship events, national sporting organizations typically coordinate team holding camps in similar environmental conditions to those expected for the championship . PreparaNutritional strategies that improve hydration status and modify body composition can support athletes’ performance in hot, humid conditions . The addThe aim of this study was to provide a comprehensive account of the preparation of elite endurance athletes during team holding camps, prior to international championship events held in hot, humid conditions. It provides specific documentation of the strategies undertaken by elite Australian male and female race walkers for the Tokyo 2021 Olympic Games: (1) baseline physiological data, training data and heat acclimation/acclimatization practices; (2) cooling strategies and nutritional strategies, and (3) Olympic Games race performance.An observational study was conducted during the holding camp for the Australian athletics team  prior to the Tokyo 2021 Olympic Games. Physiological data, training data, details of heat acclimation/acclimatization practices, cooling and nutritional intervention strategies and Olympic Games performance outcomes were documented . The teaData were collected for six elite-level athletes  of active heat acclimation sessions were gradually increased across the 7 weeks prior to the holding camp from 60 to 75 min, 32–36°C and 60–70% RH, and treadmill speed was gradually increased from 10 to 13.4 km⋅h–1. Training session pace was recorded (h) for long duration sessions and key sessions, and distances were recorded for high intensity and low intensity sessions. For each training week, intensity was expressed as the percentage of training volume (km) >4 mmol⋅L–1 blood lactate concentration , within their home training environment. For one of the female athletes, the duration (min), temperature (°C), relative humidity (% RH) and intensity  and oxygen consumption  were measured , mass using body weight scales  and sum of 7 skinfolds using skinfold calipers . An incremental, racewalking-specific treadmill test  was performed, and submaximal economy data, quantified at 11 km⋅hprotocol . Blood lmeasured .n = 6) completed 18 days’ heat acclimatization training during the team holding camp  consistent with previously reported glycerol doses ; and body mass (kg) were recorded 180 min prior to ingestion, post-ingestion and immediately post-training. Gastrointestinal (GI) symptoms , a 5-day low energy availability (EA) intervention was implemented and supervised by the team sports dietitians. The two athletes who used the LEA strategy had previously implemented similar strategies (managed by experienced sports dietitians) to achieve an ideal race body composition as part of their preparation for major international events. The daily EA target was set at 15–20 kg FFM–1⋅day–1 based upon the athlete’s training demands for each day, with additional nutrition strategies implemented, based on those recently established for endurance events in athletics . As such, athletes were actively involved in the decision-making process that led to the development of their individual race day interventions and therefore did not provide data to describe their overall perceptions or feedback of their race day interventions upon completion of this observational study.Data were reported using descriptive statistics (mean ± SD). Olympic Games race performances were reported, and the percentage change compared with the athlete’s personal best time, as documented within the World Athletics database , was cal2max result for female athletes was 63.6 mL⋅kg–1⋅min–1 and 73.7 mL⋅kg–1⋅min–1 for male athletes. The best outcome for race walking economy, defined as the lowest proportional oxygen use at 11 km⋅h–1 for females and 13 km⋅h–1 for males  was 17.5 km⋅h–1 for male athletes and 15.7 km⋅h–1 for female athletes The best 4 mmol⋅L–1 blood lactate walking speed was 80.2% vVO2max for female athletes and 86.7% vVO2max for male athletes. Training volume for the six athletes was the highest in the second week of the team holding camp, and training intensity was lowest in the first week of the team holding camp.The highest VOor males , was 61.The highest post-exercise plasma osmolality observed was recorded for one athlete who did not complete the session (athlete 4), and the athlete with the lowest post-exercise body mass change (%) in comparison with pre-exercise data (athlete 3) completed a session that was lower in volume and intensity than the other athletes . MinimalPerformance outcomes for the athletes within this study included 6th place in the women’s 20 km event (1:30:39), which was the athlete’s second-best performance time of all 20 km races within her career, and 8th in the men’s 50 km event (3:52:01), which was a personal best performance time. For the athlete who achieved 6th place in the 20 km event, her performance in the Olympic Games was a 2.0% performance decrement compared with her personal best performance (1:28:50). Additionally, one of the athletes achieved a personal best performance in the men’s 20 km event (1:24:00); This observational study provides novel data on the specific preparatory strategies of elite male and female endurance athletes competing in hot, humid conditions at the 2021 Tokyo Olympic Games. The athletes in this study utilized a combined approach of passive and active heat acclimation training prior to embarking on heat acclimatization training during the team holding camp, as well as intervention strategies targeting hydration, cooling, and body composition manipulation. This study combined the collection of both observational and self-report data and documents a careful integration of a broad range of strategies to support optimal performance in challenging environmental conditions.The training volume completed within the team holding camp for the athletes within this observational study was similar to that previously reported for elite female and male race walkers  during tThe athletes in this observational study utilized a combined active and passive intermittent heat acclimation approach, over 6–7 weeks. As an example, one of the female athletes completed 13 passive and 14 active sessions over 7 weeks. Although this strategy was not strictly consistent with recent recommendations suggesting a minimum 6–7 consecutive days of heat exposure training, or an optimal acclimatization period of ∼14 days , the athHydration measures collected within the current study indicated that the increase in plasma osmolality was similar in magnitude to that previously reported after ingestion of hyperhydration agents , and the2max, exercise economy, and cooling capacity, all of which may be associated with enhanced performance in endurance events . Additionally, we unfortunately obtained erroneous core temperature data from core pills, thereby limiting any inferences in the athlete’s heat acclimatization status. These data could have provided further insight into the ability of elite endurance athletes to undergo adaptations to heat exposure training prior to an international event. However, the findings of the current study provide some important practical recommendations for elite endurance athletes preparing for events in hot and humid conditions. Systematically planned and periodized heat acclimation over a period of 6–7 weeks, using a combination of passive and active sessions in an intermittent fashion may allow athletes to prepare for more challenging heat acclimatization training sessions leading into holding camps or pre-event training blocks that utilize the continuous HA approach. The combination of hyperhydration with internal and external cooling prior to key training sessions in the heat might allow athletes to simulate expected race day conditions and pacing strategies. It should be noted, however, that the athletes in this study refined their heat acclimation and acclimatization, cooling and hyperhydration strategies, under the management of coaches, sport scientists and dietitians, over a period of ∼2–3 years, facilitating the development of a combination of heat mitigation strategies that were tailored to athletes’ specific needs . This individualized approach culminated in well-established routines that were implemented for each athlete during the holding camp and Olympic Games.This study is one of the first to comprehensively detail the preparatory strategies of elite endurance athletes for competition in hot and humid conditions at an Olympic Games. Nevertheless, several limitations are noted including the small sample size. However, two of the six finished within the top eight within their respective events, and therefore this study may provide some insight into the individual preparation strategies of high-performance athletes. Furthermore, the quantification of internal measures such as heart rate was not included within the current investigation, as the athletes within the observational study do not typically wear heart rate monitors during training or racing. Rather, training for this group of athletes is typically prescribed using external measures including training volume (km) and speed  and passive heat exposure (1–2 sessions per week) 6–7 weeks prior to a heat acclimatization training period, ideally conducted concurrently with a team holding camp utilizing continuous HA training. On the day of key training sessions and major races, cooling strategies and hydration/hyperhydration strategies should be implemented prior to and during races, as additional heat mitigation strategies. A periodized nutrition strategy can be implemented, under the guidance of experienced sports dietitians, to modify body composition, as part of a comprehensive, periodized preparation for championship races held in hot, humid conditions.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 the Central Queensland University Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.AC, BV, JR, AR, LB, and JG contributed to the study design, data collection, data analysis, and manuscript preparation and revision. All authors agreed to be accountable for the content of the work.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.


Adolescence is a critical period of development with increased sensitivity toward psychological stressors. Many psychiatric conditions emerge during adolescence and animal studies have shown that that acute stress has long-term effects on hypothalamic-pituitary-adrenal axis function and behavior. We recently demonstrated that acute stress produces long-term electrophysiological changes in locus coeruleus and long-lasting anxiety-like behavior in adolescent male rats. Based on prior reports of increased stress sensitivity during adolescence and increased sensitivity of female locus coeruleus toward corticotropin releasing factor, we hypothesized that the same acute stressor would cause different behavioral and physiological responses in adolescent female and adult male and female rats one week after stressor exposure. In this study, we assessed age and sex differences in how an acute psychological stressor affects corticosterone release, anxiety-like behavior, and locus coeruleus physiology at short- and long-term intervals. All groups of animals except adult female responded to stress with elevated corticosterone levels at the acute time point. One week after stressor exposure, adolescent females showed decreased firing of locus coeruleus neurons upon current injection and increased exploratory behavior compared to controls. The results were in direct contrast to changes observed in adolescent males, which showed increased anxiety-like behavior and increased spontaneous and induced firing in locus coeruleus neurons a week after stressor exposure. Adult males and females were both behaviorally and electrophysiologically resilient to the long-term effects of acute stress. Therefore, there may be a normal developmental trajectory for locus coeruleus neurons which promotes stress resilience in adults, but stressor exposure during adolescence perturbs their function. Furthermore, while locus coeruleus neurons are more sensitive to stressor exposure during adolescence, the effect varies between adolescent males and females. These findings suggest that endocrine, behavioral, and physiological responses to stress vary among animals of different age and sex, and therefore these variables should be taken into account when selecting models and designing experiments to investigate the effects of stress. These differences in animals may also allude to age and sex differences in the prevalence of various psychiatric illnesses within the human population. Adolescence is an important period of transition between childhood and adulthood and is a sensitive period for stressor exposure . NeuroenBehaviorally, adolescence in humans is characterized by a pursuit of independence, changing social status, and increased risk-taking behavior. These changes are paralleled in rodents by increased play behavior and social interactions, and greater exploration of novel environments . It has A major brain nucleus involved in the modulation of the central stress response is the locus coeruleus (LC), which exerts its actions through the release of the neuromodulator norepinephrine (NE). It is activated by stress through release of corticotropin releasing factor (CRF), which interacts with corticotropin releasing factor receptor 1 (CRFR1). This increases tonic firing of LC neurons and promotes forebrain release of NE . While tWe have previously shown that a single acute stressor exposure  is sufficient to produce behavioral and LC electrophysiological changes a week after stress in adolescent male rats ,2020. Hoad libitum. Animals were handled prior to and during experimentation to habituate to the researcher. Animal protocols were approved by the Rowan University Institutional Animal Care and Use Committee and were conducted in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals.Adolescent (30–35 PND) male and female, and adult (77–82 PND) virgin male and female Sprague Dawley rats , were housed two to three per cage on a 12 h reverse light schedule (lights on at 9:00 pm) with access to standard rat chow and water Rats underwent stessor exposure or control conditions as previously described ,2020. BrIn some of the animals used for behavioral and electrophysiological experiments, blood (0.2–0.4 mL) was collected from the saphenous vein immediately before the onset of stress or control conditions (labeled as “baseline”) and 35 min after the onset (labeled as “35 min”). In subjects who were studied for long-term effects, in addition to the baseline and acute collections, blood was also collected from the saphenous vein after behavioral testing one week after control or stress conditions and was labeled as “1 week”. In all cases, blood was collected per the protocol for frequent blood collection from the lateral saphenous vein in unanesthetized animals  into 1.5Vaginal swabs were taken regularly from adult female rats throughout the study (4–5 days in a row followed by 2–3 days off) in order to effectively track each animal’s estrous cycle. Females were swabbed at the onset of the dark cycle (∼9 am–10 am) the weeks before electrophysiology recordings and approximately 30 min before the animals were euthanized for recordings to identify the estrous stages of the rats on the days of stressor exposure and the days of behavior. Vaginal samples were collected by gently swabbing the vaginal canal using a saline-dipped cotton-tipped swab and applying to glass microscope slides. Slides were stained with toluidine blue and examined using light microscopy. Estrous cycle stage was determined by the presence and morphology of cells based on previously published criteria by other laboratories . Each saImmediately after exposure to stress or control conditions, rats were placed in the center of an elevated plus maze (EPM). The EPM consisted of a plus shaped black plexiglass apparatus elevated 76 cm off the ground with two sets of opposing arms (each arm = 40cm in length) meeting in a central 10 cm × 10 cm area. Two opposing arms have vertical walls extending 30 cm from the floor of the maze, while the other two arms do not have walls. Rats were allowed to explore the maze for 10 min, during which their activity was filmed with an infrared camera. At the conclusion of each test, rats were returned to their home cage for a week. Behavior was scored using AnyMaze behavioral tracking software .One week after testing in the EPM, rats were placed in the center of an open field test (OFT) apparatus. The OFT apparatus consisted of a 90 cm × 90 cm × 30 cm black plexiglass box. Rats were allowed to explore the apparatus for 10 min, during which their activity was filmed with an infrared camera situated above the maze. At the conclusion of each test, rats were sacrificed for electrophysiological recordings. Behavior was scored using AnyMaze behavioral tracking software . Rats were tested in different mazes at the two time points to eliminate the possibility of habituation to any one test confounding anxiety-like behavior.2 2.4, NaH2PO4 1.2, MgCl2 1.3, NaHCO3, D-glucose 25. Rats were then rapidly decapitated and the skull was removed so that gross coronal cuts could be made at the level of the medulla and the pineal gland. The blocked brain was submerged in the sucrose-aCSF for 1–2 min after which it was transferred to a piece of filter paper. The dorsal aspect of the brain was cut at 200 μM thick horizontal sections using Compresstome VF-300-0Z tissue slicer. Sections containing LC were transferred into aCSF of the following composition, in mM: NaCl 126, KCl 2.5, CaCl2 2.4, NaH2PO4 1.2, MgCl2 1.3, NaHCO3 25, D-glucose 11 and continuously bubbled with 95% O2/5% CO2 and maintained at 36.0°C. After 1 h, the holding incubator was maintained at room temperature.Acute brain slices were prepared as previously described ,2020. Br2 2, EGTA 0.2, HEPES 10, Na2ATP 2, and 0.15% biocytin. After a GΩ seal was established between the pipette and neuronal membrane, the membrane was ruptured and whole-cell recordings were obtained. To assess membrane properties in current clamp mode, spontaneous activity was recorded for 60 s after stabilization of cell firing and the average firing rate was calculated. Cells were then subjected to a series of increasing current steps from -250 to 300 pA with 50 pA intervals between sweeps, and the input resistance and number of action potentials fired in response to each level of current was determined. General exclusion criteria for recordings were input resistance <100 MΩ during evoked firing and/or more than 6 Hz spontaneous firing. Otherwise both spontaneously active and “silent” neurons were included.Slices were individually transferred to a recording chamber which was continuously superfused with oxygenated aCSF and maintained at 37°C by a Warner Instrument Corporation in-line heater (model 60-01013). LC was visualized as a semi-translucent, crescent-shaped region located lateral to the fourth ventricle at 10× magnification using an Olympus BX51WI fixed-stage upright microscope with differential interference contrast and an infrared filter. Individual LC neurons were visualized with a 40× immersion lens and QImaging Rolera Bolt camera using QCapture Pro software. Neurons were approached with sharp glass electrodes (resistance = 5–10 MΩ) controlled with Sutter MPC-200 manipulators. Electrodes were filled with intracellular solution of the following composition, in mM: KCl 20, K-gluconate 120, MgClt-test was performed. If variances were unequal, a Welch modification of the t-test was conducted. Wilcoxon Rank Sum test (Mann–Whitney U test) was used for non-normally distributed data sets. Within each group  a 2  × 2  ANOVA was used to assess for differences in corticosterone concentrations. Planned comparisons with Bonferroni corrections applied to control for multiple contrasts between the following groups were used: (1) control: baseline vs 35 min; (2) control: baseline vs 1 week; (3) stress: baseline vs 35 min; (4) stress: baseline vs 1 week; (5) 35 min: control vs stress; and (6) 1 week: control vs stress. For adolescent males, tests of the significance of the Pearson correlation coefficient were used to explore relationships between various behavioral and physiological parameters. Statistical significance was set to alpha = 0.05 in all analyses. Error bars in all figures are presented as mean ± SEM.Electrophysiological data were recorded using Molecular Devices ClampEx 10.2 and MultiClamp 700B acquisition software and analyzed with ClampFit 10.2. Behavioral data were acquired and analyzed using AnyMaze behavioral tracking software . Statistical analyses were performed with GraphPad Prism Version 8.0.1. Data was tested for normality using Shapiro–Wilk test. Normally distributed data were tested for equal variances of distribution, and, for the data with equal variances, two-tailed t = 4.409, df = 11, p = 0.0060), along with a significant difference between baseline and 35 min time points exclusively in the stress group .To demonstrate the effects of acute stress on adolescent female rats, the study was performed according to the experimental timeline shown in t = 0.7003, df = 18, p = 0.4927). However, stressed female rats spent significantly more time in the center of the OFT a week after stress . There was also no effect of stress on distance traveled in the EPM immediately after stress  or in the OFT a week after stress .Anxiety-like behavior was assessed immediately after  and one p = 0.0819, U = 807.5; t = 0.6814, df = 89, p = 0.4974, U = 782.5, p = 0.0360), 100 pA , 150 pA , 200 pA , 250 pA , 300 pA .There was a non-significant trend toward a decrease in spontaneous firing rate of female LC neurons one week after stressor exposure compared to controls , 100 pA , 150 pA  and 250 pA current injection  shows that distance traveled in the EPM (Previous findings from our lab have shown that a single episode of combined restraint and predator odor stress in adolescent male rats leads to specific changes in LC physiology and anxiety-like behavior ,2020. HeThe HPA axis is the central regulator of the stress response. Its activation results in the release of steroid hormones by the adrenal glands, which leads to energy metabolism mobilization in order to adapt to an adverse situation . We obseThese data also match well with findings from other studies which have shown that adolescent female and male rats differ in their stress response. For example, both male and female Wistar rats display reduced anxiety-like behavior in adulthood after acute lipopolysaccharide-induced inflammatory stress during adolescence, but acute and chronic restraint stress during adolescence reduced anxiety-like behavior only in females . AdolescThe fact that there are disparities within and between male and female rats with regards to how they respond to stress may be explained in part by the variable nature and the duration of stressors used in the literature. Furthermore, while the behavioral effects of stress in both sexes have been extensively explored, there are few studies of sex differences in stress responsiveness of the LC/NE system in adolescent rats, despite its sexually dimorphic nature and a clear role in mediating arousal, vigilance, and anxiety-like behavior. Therefore, we assessed electrophysiological characteristics of LC neurons using whole-cell patch clamp electrophysiology. We found that, in contrast to LC neurons from adolescent male rats, which become more excitable following stressor exposure, those from stressed adolescent females are less excitable in response to current injection compared to control LC neurons. This was a surprising finding because the female LC/NE system is known to be more susceptible to CRF and has been demonstrated to be more sensitive to both acute hypotensive and swim stress . Such deAnother relevant question that this study raises is why adolescent male and female rats show opposing changes in anxiety-like behavior and LC neuronal physiology in response to stress, while human females are generally more susceptible to stressor exposure and have almost twice as high of an incidence of psychiatric conditions such as PTSD , anxietyWhile we observed opposing stress-induced changes in LC physiology and anxiety-like behavior between adolescent male and female rats, any such changes in adult animals regardless of sex were notably absent. This is of particular interest because in humans, adolescence is a critical period of development when many psychiatric disorders first emerge due to structural and functional synaptic changes in the CNS . Our datin vivo electrophysiology recordings after social stress (While this study investigated stress-induced changes in LC physiology and anxiety-like behavior occurred within groups of rats of specific ages and sexes, it did not explore any such differences between groups. This was done in order to preserve statistical power to detect effects within groups; however, others  have repl stress , where al stress .The findings from these studies indicate that behavioral and LC neuronal responses to stressor exposure differ according to age and sex. These findings highlight the necessity to consider age and sex as important sources of variation when designing and performing experiments which investigate stress, behavior, and LC physiology. There are multiple factors involved in the development of psychiatric disorders such as anxiety disorders or PTSD, which include different brain areas and neurotransmitter systems, genetic predispositions and genetic polymorphisms, and emotional and behavioral coping strategies. While LC is only one of many systems that contributes to behavioral state and the stress response, these findings help to clarify how it contributes to these phenomena among rats of different age and sex. These disparities may further allude to important differences between male and female humans of different ages in the prevalence, progression, and pathophysiology of various clinical psychiatric conditions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Rowan University SOM IACUC.OB and JT: conceptualization, data curation, formal analysis, investigation, methodology, project administration, supervision, validation, visualization, roles/writing—original draft, and writing—review and editing. CC: data curation, formal analysis, investigation, methodology, validation, visualization. JL: conceptualization, project administration, and supervision. DC: conceptualization, funding acquisition, project administration, resources, supervision, validation, and writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


S. fuscescens, a candidate species for aquaculture development in Asia. Here, to assess whether the immunostimulatory effect was facilitated by changes to the gut microbiome, we investigated the effects of those same seaweed species and four commercial feed supplements currently used in aquaculture on the bacterial communities in the hindgut of the fish. Since we found no correlations between the relative abundance of any particular taxa and the fish enhanced innate immune responses, we hypothesised that S. fuscescens might have a core microbiome that is robust to dietary manipulation. Two recently published studies describing the bacteria within the hindgut of S. fuscescens provided an opportunity to test this hypothesis and to compare our samples to those from geographically distinct populations. We found that, although hindgut bacterial communities were clearly and significantly distinguishable between studies and populations, a substantial proportion (55 of 174 taxa) were consistently detected across all populations. Our data suggest that the importance of gut microbiota to animal health and the extent to which they can be influenced by dietary manipulations might be species-specific or related to an animals’ trophic level.We recently demonstrated that dietary supplementation with seaweed leads to dramatic improvements in immune responses in  Changes in the communities of microbiota within the gastro-intestinal tracts of fish (their ‘gut microbiomes’) have recently been linked to impacts on fish health and condition, including their metabolism, overall size, and immune responses . This emAquaculture recently replaced wild fisheries as the main source of seafood globally  and its Diet can strongly influence the structure of gut microbiomes in some fish ,12 and dOur recent review revealed the exciting potential of seaweeds (marine ‘plants’) as immunostimulants for farmed fish, and also highlighted major gaps in our understanding, including the potential mode/s of action of successful immunostimulants . IndeedSiganus fuscescens [S. fuscescens were correlated with shifts in the GI microbiomes of those fish and thus, whether a mode of action of dietary seaweed immunostimulation may have been microbially-mediated. Seaweeds used in this trial included members of the red, green, and brown taxonomic groups and species that produce a broad range of bioactive, natural compounds . ssum sp. ,21 demoncrobiota , suggestSiganus fuscescens) was the focus of our study because of its candidature for aquaculture development in Asia [S. fuscescens to support the development of a sustainable farming industry for this species.Rabbitfish of the Siganidae family are marine herbivores presently receiving increased attention due to their attractive attributes for aquaculture ,24,25,26S. fuscescens. Our aims here were two-fold: firstly, to provide some ecological context for our results and, secondly, to provide baseline data on the microbiome of Siganids to help facilitate the aquaculture development of this fish species.When comparing hindgut microbial communities in fish fed different diets during our experiments, we became interested in the possible existence of a core microbiome in this species. ‘Core microbiomes’ are variably defined in the literature  and remSiganus fuscescens: ranging from 15 cm/70 g to 21 cm/189 g) were captured between February and March 2018 using a drag net (15 m long by 2.1 m deep with a 2.5 cm mesh size) on rocky reefs at Moffat Beach, Queensland Australia  were evaluated as functional ingredients in feeding trials with S. fuscescens , a hydrolysed yeast culture derived from the sugarcane fermentation process (and a source of β-glucans), (ii) sodium alginate, the anionic polysaccharide extracted from brown seaweeds, (iii) the cyanobacteria spirulina  and (iv) cracked and window refractance encysted (>95%) dried biomass of the microalga Haematococcus pluvialis, which is a source of astaxanthin. Together there was a total of 15 supplement treatments in the trial. The proximate composition of each supplement was determined following the recommended methods of the Association of Official Analytical Chemists [We aimed to screen multiple species of taxonomically and chemically diverse seaweeds for their effects on the hindgut microbiomes of Chemists , with thChemists . The souS. fuscescens were produced using the commercial aquafeed ‘Native’ . The pellets  were powdered then added to a blender  with deionised water (30% weight/weight) and combined for approximately 10 min at low speed (agitator rpm of 104) using a dough hook to produce a stiff dough. The dough was extruded through a 4 mm die onto trays which were then placed in a fan-forced oven overnight at 50 °C. Once dried, the feed was packaged in airtight bags and stored at 4 °C until required. All 15 experimental diets (supplemented with seaweed or aquafeed supplement) were made in the same manner but received supplements at 3% dietary inclusion which were powdered and sieved through a 300 μm mesh prior to the addition of water during the blending step. The use of an unsupplemented, control diet is standard practice in aquaculture to test the effect that specific ingredients may have on fish [For the preparation of the seaweed-supplemented diets, fresh seaweeds were rinsed with saltwater 34.5 ppt) to remove sand and biological contaminants. They were then spun in a washing machine  on spin cycle (1000 rpm) for 5 min to remove excess water, frozen at −80 °C, and then lyophilised in a freeze dryer  for 3 days at approximately −44 °C and 206 mbar. Once dried, each seaweed species was vacuum-sealed in individual bags with silica desiccant and stored at −20 °C until used. The ‘control’ (unsupplemented) diets for experimental  on fish ,33,34. T.5 ppt ton = 3) to have a total of 48 tanks, all of which were then stocked with three fish each . Due to variation in sizes, fish (N = 144) were sorted into two size classes: ‘small’ (ranging from 15 cm/70.5 g to 18 cm/112.1 g) or ‘large’ (ranging from 18 cm/112.4 g to 21 cm/189.2 g). The 144 fish were randomly allocated into 48 tanks so that each replicate tank contained 3 fish with at least one small and one large fish. The exact mass and length of each fish were recorded and used as a covariate in analyses assessing the influence of fish diet on microbial community diversity. As processing limitations were forecasted for the end of the trial, the fish were stocked in a staggered manner with one tank per treatment stocked each day over three days to allow for the sampling of one tank per treatment each day over three days at the end of the screening trial.This study used three replicate plastic tanks (55 L) for each of the 16 dietary treatments . Therefore ‘Tank’ was a random factor nested within the fixed factor of ‘Diet’. To enable staggered sampling at the end of the experiment and ensure that all fish were exposed to the treatment diets for the same time period (two weeks), one out of three tanks from each dietary treatment was stocked with fish each day, over three days. The 3/h generator units . The ozone-treated seawater was then pumped via ultraviolet filters, providing 80 mJ/cm2, to two 4 m × 2.2 m granular activated carbon vessels for a contact time of >9 min to remove unwanted by-products from the ozone treatment. Finally, the seawater was pumped to a header tank, which fed directly into a pipe system delivering treated seawater to this experiment. The system was maintained in a temperature and light controlled room kept at 24–26 °C and on a 24L:0D dim central light regime.Fish were fed by hand at 3% body weight twice a day (10:00 and 15:00) for a period of 14 days. The reason for the trial lasting 14 days is based on our previous review [® , then the entire digestive tract from each fish was aseptically excised and placed in a Falcon tube (50 mL) before being snap-frozen and stored at −80 °C until further processing could occur.After the feeding trial (14 days), the fish were subjected to a 24 h fasting period. The fish were euthanized in 10 ppt Aqui-SThe full methodological details of samples obtained for analysis of innate immune responses to the different seaweed diets are described in Thépot, et al. . BrieflyUlva fasciata treatment which only had 2 tanks). After the samples thawed, 0.25 g (approximately 0.5 cm length) of hindgut containing digesta was sampled. Our rationale for choosing to sample the hindgut with digesta was based on results published by Nielsen, et al. [To compare the hindgut microbiomes of fish fed different experimental diets, DNA was isolated from the hindgut and digesta of one randomly selected small and large fish from each tank  [https://www.bioconductor.org/packages/release/bioc/html/dada2.html, accessed on 10 January 2021) [https://www.arb-silva.de/, accessed on 10 January 2021) [From the isolated DNA, the 16S rRNA gene was amplified using PCR following previously published methods ,36,37. Bry 2021) . To ensury 2021) , using try 2021) . Trimmedry 2021)  and QIIMry 2021) . Due to Laurencia treatment  and the removal of 52 ASVs no longer present after rarefaction, leaving a total of 1,198 ASVs and 47 samples.Approximately 95.1%  of total reads were quality filtered and retained through this process. Subsequent quality filtering included the removal of singletons, chimeric sequences, mitochondrial DNA, and unassigned or eukaryotic ASVs. This resulted in a total of 1250 ASVs from 48 samples. Rarefaction to 6290 counts was performed to account for uneven sequencing depth among samples  . This ren = 9 fish, 3 fish per tank). Here a subset of these fish per tank (n = 3) were randomly selected and are then related back to the immune data of those individual fish. These were included in MDS and PERMANOVA as per the Data Analysis (below).The recently published paper  characteS. fuscescens. With the permission of those authors and the provision of raw sequence data from those papers, we compared the microbiomes of our captive fish  to the results obtained from fish caught from wild populations on the east and western coastlines of Australia, respectively. Nielsen, et al. [S. fuscescens captured nearby One Tree Island  in the tropical Great Barrier Reef (GBR), whereas Jones, et al. [Two recently published papers ,29 also , et al.  charactehttps://github.com/ncbi/sra-tools, accessed on 10 January 2021) and processed as demultiplexed fastq files. Raw data from both comparison studies (sequenced in the V4 region) were also processed using the cutadapt package [Laurencia treatment;  and the removal of 76 ASVs no longer present after rarefaction, leaving a total of 3084 ASVs and 85 samples  [S. fuscescens fed the different diets was explored in a non-metric multidimensional scaling (NMDS) using Euclidian distance and compared between treatments using PERMANOVA. Differences between means are considered significant at p < 0.05. Alpha diversity of microbial communities, Observed ASVs, and Shannon-Weaver index (hereafter “Shannon index”), were compared among fish fed different diets and later, between different studies, using Kruskal-Wallis tests. For the rest of the analyses, to allow the comparison of both sequencing runs on a shared number of ASVs, the rarefied ASVs were agglomerated at the genus level. Venn diagrams were used to show the number of shared ASVs among samples and studies and were constructed using the Limma package [After processing, data were imported into R v3.6.3 (available at er 2020)  using ther 2020)  for stat package . Beta di package . ASV levS. fuscescens, the 47 fish fed the different treatment diets in our trial were combined under the “Sunshine Coast” population. The four geographically distinct rabbitfish populations were analysed using pairwise comparisons of changes in the relative abundances of raw, un-rarefied data using Wald tests in the DESeq2 function [p-values were adjusted using the Benjamini and Hochberg method. ASV level differences between each population  were evaluated using multiple one-way ANOVAs, with square root transformed data to meet the assumptions of homogeneity of variance and improve normality. Significant ANOVA (p < 0.05) results were followed by a Tukey’s HSD post hoc test. Additionally, the package microbiome [In order to compare our fish as one population (“Sunshine Coast”) to the other 3 wild populations of crobiome  was usedcrobiome ,50, to icrobiome ), of samF = 0.39, p = 0.846; Firmicutes; Arcobacter sp.), which was a highly abundant taxon in the hindgut of the fish fed the control diet  used in our experiment. To allow the comparison of both sequencing runs from this trial at the ASV level, the rarefied ASVs abundance agglomerated at the genus level. This left 113 assigned genera in total from all 5 treatment groups . Out of these 113 taxa, the hindgut of the fish fed the control and supplemented diets shared 63 taxa  did not show clear differences between the composition of hindgut microbial communities in fish fed different diets compared to the control fish (p = 0.104 and length: p = 0.15 for unweighted UniFrac measure).The beta diversity results  was represented by just three taxa and accounted for an average 44.0% ± 1.42% relative abundance across our samples (Mean ± SE). The most abundant taxon was the genus Desulfovibrio and represented between 11%  and 34% , with the lowest relative abundance observed in the hindgut of fish fed the control diet (9.3% ± 4.0%) compared to an average of 21.1% ± 1.4% for all other treatments  of Firmicutes and the average value for the seaweed supplements was 20.5% ± 1.7%. Conversely, fish on the control diet seemed to possess a higher proportion of bacteria in their hindgut belonging to the phylum Epsilonbacteraeota  which was lower in fish fed control diets compared to those fed supplemented diets, with the highest relative abundance for that taxon observed in fish fed the calcified green seaweed Halimeda sp. . The relative abundance of bacteria from the Arcobacteraceae family also appeared to be higher in hindguts of fish fed control diets , it had extremely low abundance (0.3% ± 0.2%) in the hindgut of fish fed the control diet , we did find some differences between the communities in the hindgut of fish fed any of the supplemented diets compared to those fed the control diet at the genus level. For example, although the relative abundance of S. fuscescens from the three combined studies  from Jones, et al. [To provide ecological context for our results and additional information for the development of bespoke aquafeed for this fish species, we compared our microbiome data, obtained from fish collected on the subtropical Sunshine Coast, Australia, in 2018, to those obtained from conspecific hindgut microbiomes from individuals in populations located ~4000 km west off the Western Australian (WA) coastline (Shark Bay and the Kimberley Coast) and ~350 km north on the Great Barrier Reef  during 2015 and 2016. In total, we recovered 3084 ASVs after rarefaction  and the Shannon index: F = 85.81, p = 0.12) of S. fuscescens  were also significantly different from each other  but not on UniFrac .The analyses of beta diversity revealed strong differences between the four populations of Proteobacteria (52.6% ± 2.9%) compared to all the other fish including those in our study (30.9% ± 2.2%) and the fish from WA (26.5% ± 1.9% and 20.6 % ± 2.0% for Shark Bay and the Kimberley fish respectively), however, this was not resolved statistically , and they also had the lowest relative abundance of Fusobacteria  and Epsilonbacteraeota , which across all the other fish represented an average of 3.8% relative abundance  but it was significantly lower in those from the Kimberley  and the GBR , significantly higher than for GBR fish (Tukey ‘Sunshine Coast vs GBR’ adjusted p = 0.0.24).Fish from Western Australia had more similar hindgut microbiomes to the fish from our feeding trial compared to those from the GBR. For example, the relative abundance of Spirochaetes  in the GBR and WA fish, compared to our samples which had a relative abundance of 5.7% ± 0.5%  in abundance between the hindgut microbiome of one or more of the four geographically distinct populations of rabbitfish from the 3 studies  the dietary supplementations (3% dietary inclusion) which led to profound immune responses in the fish were insufficient to elicit a strong change in the fish GI microbiomes, (ii) the immune response we observed in experimental fish was unlikely to have been microbially mediated, (iii) there is potential for the existence of a stable, core GI microbiome in S. fuscescens, for which we conducted a further investigation with published data from two other studies [Supplementation of diets with seaweeds and commercially available aquafeed supplements had only subtle effects on the diversity and composition of hindgut microbial communities in the rabbitfish eviously  showed a studies ,29.S. fuscescens appears surprisingly stable, despite experimental manipulations of diet at levels that are known to be able to fundamentally change the outcomes of production and other fish traits [Despite some clear and expected differences between the GI microbiomes in fish from our study and the two other studies, 55 out of 174 assigned taxa were shared, and 13 of those represented between 66% and 85% of the total relative abundance in all fish. These observations from geographically and temporally distinct populations—including our fish which were all fed experimental diets based upon commercial fish pellets developed for carnivorous fish (with and without seaweed and other supplements)—provide compelling, initial evidence for a possible core microbiome in this opportunistic omnivorous subtropical fish species. The hindgut microbiome of h traits .Aeromonas hydrophila [S. fuscescens  were noAsparagopsis taxiformis on the immune responses in S. fuscescens, were direct and not mediated by microbiomes in the hindgut of the fish. However, it is possible that the seaweed dietary supplements had effects on the microbiomes of the fish outside of their hindgut, including the skin and gills, which have previously been reported to be locations where the microbial community can be influenced by diet [These observations suggest that the effects of seaweeds, especially  by diet . Further by diet . It is p by diet ,52. FutuSparus aurata and Seriola lalandi [Oncorhynchus mykiss and Siganus canaliculatus [Siganus canaliculatus , iculatus ,33,60). iculatus  found thscescens ) with 10reatment . Out of reatment , three, Asparagopsis taxiformis, Caulerpa taxifolia, and Sargassum spp.). Indeed, there was some evidence for the effects of these chemically ‘rich’ seaweed supplements. For example, fish fed diets supplemented with the green alga Caulerpa taxifolia had significantly higher levels of Fusobacterium spp. and similarly enhanced Cetobacterium spp. and Treponema spp. C. taxifolia produces many interesting bioactive compounds [Girella tricuspidata) and invertebrate grazers in Australia [A. taxiformis was also expected to significantly modulate the GI microbiome of the rabbitfish fed that supplement due to its production and storage of potent antimicrobial bioactives [Romboutsia sp.) and these changes were not statistically significant.Although seaweed is a highly diverse group  that produce a wide range of secondary metabolites with bioactive properties and are increasingly used as animal supplements, there is a gap in the literature regarding their potential as a dietary supplement to shape the intestinal microbiome of animals including fish ,63. Oursompounds  and its ompounds  and refeustralia  and can ustralia ,66. The oactives  and its oactives ,68. HoweS. fuscescens that were sampled by different teams at different times from different places had significantly different hindgut microbiomes. However, there was still substantial overlap, with almost 18% of bacterial taxa present across all populations. Interestingly the hindgut microbiome of our fish appeared to have more in common with that of the fish from Western Australia (~4000 km away) than with the geographically closer population of S. fuscescens from the GBR (~380 km away), which seemed more distinct than other populations. Overall, the hindgut microbiome composition of the fish from our study (Sunshine Coast) was most similar to those from Shark Bay in Western Australia, which is at a similar latitude.Not surprisingly, the geographically distinct populations of Siganus guttatus, across three sites separated by up to 390 km across a three-year sampling program [Siganus spp. have a core microbiome in their GI tracts [Potential explanations for these groupings are that all of the seaweed genera fed to our fish have tropical, subtropical, or temperate distributions and are common on the eastern coast of Australia, with many also occurring on the west . It is,  program . This obI tracts ,26,34,70Siganus species may have a core microbiome that is robust to dietary manipulations and large geographical distances as per other Siganus spp. [S. fuscescens does indeed have a core microbiome and importantly, the functional roles of any core microbial taxa throughout the life of Siganus fish in the wild and on farms.Despite experimental manipulations of diets with taxonomically and chemically diverse seaweeds and samples originating from populations in locations separated by up to 4000 km around the Australian coast  and commercial products. These results are contrary to emerging studies from other fish species and animals-including humans-and suggest that the effects of diet and functional feeds on animal gut microbiomes and resulting health may be species-specific and influenced by trophic levels. Our observations provide some preliminary evidence that a conserved core microbiome may exist within the hindgut of this fish species and provide other baseline data about temporal and spatial variation in the hindgut bacterial communities within this candidate aquaculture species, which may support the sustainable development of this industry.Immunostimulatory effects of dietary supplementation with seaweeds in the mottled rabbitfish


Drosophila melanogaster (Canton-S strain) were exposed to different concentrations  of cadmium. Trypan blue staining showed that intestinal cell damage of Drosophila larvae increased and the comet assay indicated significantly more DNA damage in larvae exposed to high Cd concentrations. The nitroblue tetrazolium (NBT) experiments proved that content of reactive oxygen species (ROS) increased, which indicated Cd exposure could induce oxidative stress. In addition, the expression of mitochondrial adenine nucleotide transferase coding gene (sesB and Ant2) and apoptosis related genes  changed, which may lead to increased apoptosis. These findings confirmed the toxicity effects on oxidative stress and cell apoptosis in Drosophila larvae after early cadmium exposure, providing insights into understanding the effects of heavy metal stress in animal development.With the increase of human activities, cadmium (Cd) pollution has become a global environmental problem affecting biological metabolism in ecosystem. Cd has a very long half-life in humans and is excreted slowly in organs, which poses a serious threat to human health. In order to better understand the toxicity effects of cadmium, third instar larvae of  Smoking is another common way of introducing Cd into the human body. Cd exposure in infants and juveniles directly affects development and growth, leading to the destruction of the testicular blood barrier, germ cell apoptosis, testicular oedema, hemorrhaging, necrosis, increased lipid peroxidation, and infertility3. Additionally, excessive Cd exposure can cause genomic instability, which has been linked to various cancers4. Thus, it is crucial to further elucidate the effects of Cd exposure, as it poses a significant risk to human health and environment.Cadmium (Cd) is not an essential element for all organisms, and its presence, even in low concentrations, can have toxic effects. In industrial settings, the inhalation or ingestion of Cd can cause acute or chronic poisoning. In humans, food is the main source of Cd exposure5, and act as signal molecules in cell differentiation, cell cycle, and cellular response to external stimuli6. Cd activates the mitogen-activated protein kinase (MAPK) pathway which is related to the transcription of oxidative stress signal molecules. Chen et al.7 and Oswald et al.8 studied the locomotive behavior of Drosophila larvae, and found that ROS acted as an obligate signal for neuronal activity in Drosophila motor neurons, and were structurally plastic at the presynaptic and postsynaptic ends.ROS is an unavoidable product of aerobic cell activity. Aerobic activity produces numerous metabolites, under the control of a series of specific systems, and exceeding the metabolite threshold accelerates the production of ROS. High level of ROS can mediate damage to cell structures, lipids and membranes9. Changes in mitochondrial membrane potential, structure, and permeability occur during apoptosis in Drosophila10. Apoptosis can maintain cellular environmental homoeostasis, and is an autonomous and spontaneous behavior that is of great significance for maintaining organism health, nervous system development, and immune system functions11. The Jun N-terminal kinase (JNK) is the main apoptosis-promoting factor in Drosophila that can activate a variety of transcription factors, such as AP-112. Dying cells produce high levels of ROS and JNK,elevated JNK levels can promote cell apoptosis or increase ROS production through other mechanisms.When Cd-induced damage exceeds the cell’s ability to repair itself, the signal transduction pathway is activated to initiate the process of apoptosis, which can minimize damage to a local areaDrosophila after complete metamorphosis and development13. Exposure to Cd at the larval stage of Aedes albopictus can reduce survival, prolong development time, and reduce the weight, reproduction, and survival rate of adults14. This may be due to the interaction of Cd with biological macromolecules of organism, which may disrupt the normal process of cell division, introduce oxidative stress from excessive ROS, or consume excessive energy with detoxification mechanisms.Cd, which enters larva through ingestion, can still be detected in Drosophila melanogaster (Canton-S strain) was used as experimental material to explore the effects of heavy metal cadmium on oxidative stress and apoptosis of third instar larvae, so as to provide a more comprehensive understanding of the toxic effects and toxicity mechanism of early cadmium exposure on Drosophila melanogaster larvae.In this study, Analytical-grade Cd chloride was purchased from Fuchen Chemical Industries Ltd. . Collagenase IV and trypan blue were purchased from Beijing Solarbio Technology Ltd. . NBT, comet assay, and DNA damage detection kits were acquired from Nanjing Jiancheng Bioengineering Institute . SYBR Green I and premix Taq DNA polymerase were obtained from Hunan Aikerui Biological Engineering Ltd. . The RNAiso Plus Kit was acquired from Beijing Baori Medical Biotechnology Ltd. .National Standard for Food Safety (GB-2762-2017) as a reference point, the median lethal concentration (LC50) was determined by different culture mediums with nine Cd concentrations: 0 mg/kg (control), 1 mg/kg, 2 mg/kg, 4 mg/kg, 8 mg/kg, 16 mg/kg, 32 mg/kg, 64 mg/kg, and 128 mg/kg.Using the maximum Cd concentration permitted by the Drosophila were collected within 8 h after eclosion and were cultured separately for three days15. They were then placed together in activated carbon culture media, and allowed to mate for 12 h. The eggs were picked and cultured in culture mediums with different cadmium concentrations. There were 175 eggs per concentration group. Eclosion rate which is the proportion of eclosed adults from larvae was calculated. The experiments were performed in triplicate and the results were averaged.Male and female 50 experiment, four cadmium concentrations  were set up for subsequent experiments. Similar to the LC50 experiment, male and female Drosophila were collected within 8 h after eclosion and cultured separately for three days. They were then crossed in fresh control medium (without Cd) or Cd mediums for 12 h. Parental adult flies were then removed, and third instar larvae developed from different medium were selected for the experiments.Based on the results of LCDrosophila larvae cells16. Larvae haemolymph cells (extracted from fifty larvae per sample) were incubated with NBT for 1 h in the incubation medium. The insoluble formazan crystals formed were solubilized by adding 150 µL 50% acetic acid followed by 5 min vortexing. The absorbance of the final solution was measured at 595 nm using a spectrophotometer.NBT reduction test was used to detect the ROS content in 17. Seventy third instar larvae were randomly selected from each treatment group, cleaned with PBS, put in 0.02% trypan blue staining solution, and incubated for 2 h with vibration. After cleaned with PBS, the samples were checked and photographed under a stereomicroscope.Trypan blue staining was used to detect cellular damage in the midgut of larvae19.Crawling experiment was used to identify the neuronal damage in the early stage of larval development. Three instar larvae were randomly selected from Cd medium with different concentrations and washed in PBS solution, then placed in the center of Petri dish containing 0.8% agarose gel. A layer of grid paper was put under the Petri dish. After the larvae adapted for 10 min, the crawling track, turning angle and number of peristalsis contractions (full anterior to posterior movement = 1 contraction) of larvae within 2 min were recorded20. Fifty third instar larvae were randomly selected from each group. The midgut was dissected under an anatomical microscope, digested in collagenase solution for fifteen minutes, and the cells were collected by centrifugation, washed with PBS and centrifuged to make cell suspension. Then the experiment was performed according to the instructions of the DNA Damage Detection Kit . Finally, the samples were checked under the fluorescence microscope  (515–560 nm wavelength).Comet assay can effectively detect and analyze the degree of DNA damage in cellssesB and Ant2) and six apoptosis-related genes  were queried using NCBI (https://www.ncbi.nlm.nih.gov/). Primer design and synthesis were carried out at the Shenggong Bioengineering Co. Ltd., Shanghai. cDNA was obtained via RNA reverse transcription, following the instructions of the PrimeScript ®RT Master Mix reverse transcription kit. The reaction system was prepared according to the instructions of the SYBR Green kit to detect the mRNA expression of the aforementioned genes.Total RNA from six third instar larvae was isolated using the RNAiso Plus Kit , according to the manu-facturer’s instructions. The coding sequence (CDS) sequences of two mitochondrial adenine nucleotide transferase (ANT) coding genes , annealing , and extension . The experiments were performed in triplicate and the results were averaged. The amplification primers are shown in Table P < 0.05). GraphPad Prism 7.0  was used to create all graphs.All the experimental data were analyzed by SPSS 20.0 statistical software  using one-way ANOVA and Tukey’s significance testing , and the tail length , tail moment (TM), olive tail moment (OTM), and tail DNA % (TD) were determined.All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.Drosophila from larvae under different Cd concentrations was presented in Fig. 50 was used as the basis for setting the cadmium concentration in the follow-up study.The eclosion rate of P < 0.01), and the ROS level increased gradually with the increase of Cd concentration. These results showed that higher concentration Cd stress could induce a significant increase in ROS content in Drosophila larvae. These results showed that the content of ROS in Drosophila larvae increased significantly under higher concentration of cadmium stress.The ROS content of blood lymphocytes of third instar larvae was shown in Fig. Drosophila larvae was higher with increasing concentrations of cadmium (data not shown). These results indicated that the midgut cells of Drosophila larvae showed cell damage under the cadmium exposure, and the degree of damage increased dose-dependently.The third instar larvae after trypan blue staining were showed in Fig. P < 0.05) and 9 mg/kg (P < 0.01) treatment groups were significantly lower than that of the control group   Fig. C. These P < 0.05 or P < 0.01). The tail length of comet of the treatment groups  increased significantly compared with the control group (P < 0.01). These results indicated that the DNA “smearing” phenomenon became more severe, and there was a higher degree of DNA damage in Drosophila larvae with the increase of cadmium concentration.The images of comet assay of midgut cells of the third instar larvae were shown in Fig. Drosophila is closely related to mitochondria. ANT protein is a transporter related to apoptosis in mitochondrial transport family. ANT is jointly encoded by Ant1 (sesB) and Ant2 in Drosophila21. Effects of Cd on the gene expression of adenine nucleotide transferase coding genes sesB and Ant2 in Drosophila larvae were shown in Fig. sesB in treatment groups of 2.25 mg/kg, 4.5 mg/kg and 9 mg/kg decreased significantly compared with the control group (P < 0.01). The expression level of Ant2 appeared to decline first, then increase and then decrease, but there was no significant difference compared with the control group (P > 0.05). These results showed that Cd exposure inhibited the expression of sesB gene in Drosophila larvae.The increase of ROS in Debcl, hid, rpr, p53) and anti-apoptotic genes  in Drosophila. As shown in Fig. Debcl, hid, rpr, and p53 . The expression of the anti-apoptotic gene Sce was significantly downregulated in the 4.5 mg/kg and 9 mg/kg treatment groups (P < 0.01)   Fig. E. The ex01) Fig. F.Figure Drosophila intestinal cell damage increased with increasing exposure to Cd. The NBT experiments proved that ROS increased after Cd treatment. As excessive ROS has been shown to induce DNA damage22, it is unsurprising that the comet assay analysis indicated significantly more DNA damage in Drosophila exposed to high Cd concentrations. Cd could interact with ROS regulators, such as glutathione, reducing the ability to eliminate ROS, indirectly increasing ROS levels and inducing oxidative stress23. Previous studies have found that heavy metal exposure caused a decrease in catalase (CAT) and superoxide dismutase (SOD) activity and an increase in MDA content in Drosophila larvae24. This leads to the accumulation of ROS in cells, which can cause oxidative stress and subsequent DNA damage.In this study, trypan blue staining showed that Drosophila eggs treated with 250 and 300 mg graphene/L resulted in a significantly reduced crawling speed25, and those treated with high concentrations of NaF showed significantly reduced crawling speed and body wall contractions19. The abnormal crawling behavior of larvae exposed to high Cd concentrations indicates that mechanical motor neurons are affected by toxic stress, which may be caused by brain coordination defects and damage to the subesophageal ganglion. The slower crawling speed may be caused by damage to the cholinergic neurons, which can result in abnormal muscle contraction.Our results indicated that larvae in the high Cd concentration groups exhibited abnormal crawling behavior compared with the control group. Similarly, other studies have found that sesB in the third instar larvae of Drosophila decreased with increasing Cd concentration. sesB primarily encodes the mitochondrial ANT protein: mitochondria are the only source of adenylate triphosphate (ATP) within the synapses, therefore, ANT is essential for the transport of ATP from the mitochondrial membrane to the cytoplasmic matrix26. DeVorkin et al.27 found that Drosophila caspase1 (Dcp-1) is located upstream of sesB, and can promote autophagy in the salivary glands, midgut, and other parts of larvae by inhibiting sesB activity. Thus, sesB was shown to be a new negative regulator of autophagic flux in the mid-stage of Drosophila development. With a decrease in sesB expression, the mitochondrial morphology changed from elongated organelles to spherical particles, causing changes in their metabolic state and possibly inducing cell apoptosis28. The remaining mitochondria maintain tissue viability through increased activity, which can lead to an increase in ROS. When sesB was inhibited, the H2O2 content increased sharply, and O2− was produced by the G3PDH active enzyme and ubiquinone on the cytoplasmic side of the inner mitochondrial membrane29. This indicates that O2− /H2O2 are created by a phosphoglyceride shuttle in the body of Drosophila, making sesB-knockout cells more sensitive to oxidative stress. sesB also regulates calcium levels within cells and mitochondria; therefore, when sesB was knocked out, calcium signaling in mitochondria was blocked30. Changes in sesB expression generally impair mitochondrial function, leading to reduce toxic metabolism, oxidative damage, and even cell death31.This study also identified that the relative expression of Debcl has been shown to affect a wide range of other genes and functions. Debcl inhibits the expression of Buffy32, a gene involved in stress- and DNA damage-induced apoptosis that is not required for normal development in Drosophila. Additionally, the tumor suppressor gene, Rbf1, interacts with Debcl to increase the amount of ROS produced by mitochondria, leading to the activation of the JNK pathway and, eventually, to apoptosis in Drosophila33. Debcl may also increase mitochondrial ROS content through glycerophosphate oxidase 134. Overexpression of Debcl in the eyes of Drosophila also leads to eye-cell apoptosis35. Based on these previous findings and the results of this study, it can be presumed that the upregulation of Debcl expression may ultimately induce apoptosis by increasing the activity of glycerophosphate oxidase 134 and the mitochondrial fission protein Drp136. ROS content may be further increased by the increase in Gpo-1 expression, caused by the upregulation of Debcl genes in the Bcl-2 family.p53 can promote DNA repair; however, when DNA damage repair is incomplete or fails, it can promote apoptosis instead37. The upregulation of p53 may additionally stimulate the upregulation of other pro-apoptotic genes, rpr and hid in the RHG family, and inhibit the expression of the anti-apoptotic gene, Diap138. Accordingly, in addition to the upregulation of p53, the expression of rpr and hid were also upregulated after Cd stress in this study, whereas the Diap1 expression was significantly downregulated. Diap1 is a key enzyme that controls cell fate, and regulates apoptosis by selectively degrading pro-apoptotic proteins or itself36, helping to prevent apoptosis caused by abnormal caspase activation. However, the protein produced by rpr and hid inhibits the expression of Diap1 and induces the release of Dronc (a caspase-9 homolog). After Dronc is released, it binds to the scaffold protein Ark (Apaf-1) and hydrolysis activates Drice, Dcp-1 (a caspase-3 homolog)37. These caspases induce cell DNA fragmentation and the formation of apoptotic bodies39. This study indicated that Cd stress induced the upregulation of pro-apoptotic genes  and the downregulation of anti-apoptotic genes (Sce and Diap1) at the mRNA level in Drosophila larvae. Changes in the expression of these genes may also change the membrane potential in the mitochondria and lead to a harmful increase in ROS content and further DNA damage.Drosophila melanogaster. The DNA ‘smearing’ phenomenon indicated DNA damage induced by cadmium exposure. Moreover, the content of reactive oxygen species increased, suggesting oxidative stress. Cadmium could also lead to behavioral changes of Drosophila larvae, such as excessive distortion, increased turning times and slower crawling speed. In addition, Cd induced changes in the expression of adenine nucleotide transferase coding gene sesB in Drosophila larvae, suggesting that Cd stress may produce more ROS by affecting the functional changes of mitochondria. The relative expression of pro-apoptotic genes Debcl, hid, rpr and p53 increased, meanwhile, anti-apoptotic genes Sce and Diap1 decreased. In conclusion, cadmium exposure may induce oxidative stress and apoptosis in Drosophila larvae by increasing the content of reactive oxygen species. Consistent with previous studies41, this study provided further evidence about the toxicity effects of early cadmium exposure on Drosophila larvae. The present study suggests that cadmium exposure induces oxidative stress and cell apoptosis, which could be one of the main mechanisms for induction of genotoxicity.Heavy metal cadmium induced damage of midgut cells of third instar larvae in


Pinellia ternata(Thunb.) Breit., Rheum palmatumL., Gentiana scabraBunge, Radix Aucklandiae and Citrus aurantium L. This approach combines pharmacokinetics analysis with pharmacodynamics evaluation for the active compounds screening, targets prediction and pathways assessing. Firstly, through pharmacokinetic evaluation and target prediction models, 83 potential compounds and 184 gastric cancer-related targets were screened out. Then, the results of network analysis suggested that the targets of PRGRC were mainly involved two aspects: apoptosis and inflammation. Finally, we verified the reliability of the above analysis at the cellular level by using naringenin and luteolin with good pharmacokinetic activity as representative compounds. Overall, we found that PRGRC could influence the development of gastric cancer from a multi-scale perspective. This study provided a new direction for analyzing the mechanism of TCM.Because of the complex etiology, the treatment of gastric cancer is a formidable challenge for contemporary medical. The current treatment method focuses on traditional surgical procedures, supplemented by other treatments. Among these other treatments, Traditional Chinese Medicine (TCM) plays an important role. Here, we used the systems pharmacology approach to reveal the potential molecular mechanism of PRGRC on gastric cancer which composes of  Due to the atypical early symptoms, about 70% of patients with gastric cancer were in the late stage of diagnosis, which significantly limited the efficacy of surgery and radiotherapy. In the past decades, conventional chemotherapy drugs such as cisplatin (DDP) and 5-fluorouracil have brought great clinical benefits to patients with advanced gastric cancer. However, due to drug resistance and cytotoxicity, the clinical efficacy gradually deteriorates2. Traditional Chinese Medicine (TCM) is a natural medicine with low toxicity, high safety and efficacy and has curative effects on gastric cancer, affecting both basic pathophysiology and syndrome characteristics. And now, it has emerged as a research hotspot3.As one of the most common malignancies of the gastrointestinal tract, gastric cancer has a high incidence and poor prognosisPinellia ternata(Thunb.) Breit., Rheum palmatumL., Gentiana scabraBunge, Radix Aucklandiae and Citrus aurantiumL. Among these five herbs, it has been reported that Pinellia ternata(Thunb.) Breit can block SGC7901 cells in S phase, thereby inhibiting tumor activity by affecting cell cycle progression5. Then, previous researches have shown that Rheum palmatumL. and Radix Aucklandiae could inhibit cancer cell metastasis and induce apoptosis by regulating the expression of apoptosis-related proteins7. Reports in the literatures indicated that Gentiana scabraBunge and Citrus aurantiumL. have been commonly applied on diseases for their anti-inflammatory, anti-tumor and anti-oxidant properties9. For PRGRC, due to its complex composition, the molecular mechanism on gastric cancer was not yet clear. Therefore, a systematic approach to explain the mechanism of TCM is urgently needed.Due to the synergistic pharmacological and pharmacokinetic effects of multiple active compounds, TCM formulas consisting of multiple herbs play a prominent role in the treatment of various diseases. Meanwhile, many herbs and formulas have been successfully used to treat gastric cancer. This article described one of these formulas—PRGRC which consists of five herbs: 10. In our previous studies, we have successfully constructed a new systems pharmacology approach that includes ADME characterization of drugs evaluation, target prediction and network analysis, which facilitated the identification of multiple mechanisms of drugs11. Through this platform, a variety of herbs including Radix Salviae miltiorrhiza, Sophora flavescens, Sedum rosea and Glycyrrhiza uralensisFisch. have been verified to have potential mechanisms in the treatment of cancer, depression and cardiovascular diseases.Recently, the development of systems pharmacology provided a new way to explore TCMs across multiple scales of complexity ranging from molecular and cellular levels to tissue and organism levels12 and drug-likeness (DL)13 properties of compounds contained in PRGRC through systems pharmacology approach to obtain potential compounds. Next, based on the integrated target prediction methods, WES14 and SysDT15, which united the biological and mathematical models, we predicted the homologous targets of the screened potential compounds. Afterwards, the obtained targets were verified by functional enrichment analysis and the relevant networks were constructed. Ultimately, the network pharmacology and gastric cancer-related signaling pathways evaluation were carried out to systematically disclose the underlying reciprocity between active compounds, active targets and pathways. Based on the above, we explored the mechanism of PRGRC in the treatment of gastric cancer. In addition, naringenin and luteolin with good pharmacokinetic activities were used as representatives of the screened potential compounds for further verification in vitro. The results dissected the multi-scale mechanism of PRGRC, which provided examples for revealing the mechanism research of TCM.Therefore, in this article, we firstly analyzed the oral bioavailability (OB)To reveal the multi-compound and multi-target features of PRGRC on gastric cancer, we collected a total of 442 compounds from TCMSP database of all five herbs in PRGRC. In order to screen for potential bioactive compounds of PRGRC, we evaluated the ADME  properties of these compounds, including OB and DL. As shown in Table 16, Caspase 3 (CASP3)17, interleukin-10 (IL-10), nitric oxide synthase, inducible (NOS2), nuclear factor kappa-B (NFκB), Cyclooxygenase 2 (COX2)18 and other proteins were involved in the immunoregulation, apoptosis and inflammatory processes of gastrointestinal diseases, which may indicate the synergistic effect of compounds and targets on gastric cancer.In order to explore the targets of compounds for PRGRC in the treatment of gastric cancer, we identified a total of 365 direct and indirect targets associated with active compounds by means of WES and SysDT algorithms. The results showed that most compounds acted on multi-targets, proving various pharmacological effects of bioactive molecules. Afterwards, we mapped the 365 targets to the CTD database for disease enrichment analysis, thereby obtaining 184 targets associated with gastric cancer  network by Cytoscape3.6.0. As shown in Fig. 20. Meanwhile, the researches indicated that luteolin (MOL75) with the highest degree could suppress inflammation-associated gene expression by blocking the activation pathway of RELA and AP-121. Gentiopicroside (MOL50)22 and Rhein (MOL18)23 have been proved to inhibit tumor development through regulating PI3K-Akt signaling pathway. And the researches indicated that beta-sitosterol (MOL05)24 acted as the activator of estrogen receptor (ESR1) and transcription factor AP-1 (JUN). By referring the relevant literatures, we found that kaempferol (MOL42) had the effect on regulating adaptive humoral immunity by managing primary B cells and T cells in vivo25 and it may reduce the intensity of inflammatory processes by inhibiting the secretion of proinflammatory cytokine TNF-α, additionally by increasing the secretion of anti-inflammatory cytokine IL-1026. Thus, these potential compounds of PRGRC acted by modulating inflammatory responses.Among the 83 potential compounds, naringenin (MOL71) and luteolin (MOL75) were known to exert anti-inflammatory effects, as well as to inhibit the proliferation of various cancer cell lines. Naringenin (MOL71) with a degree of 36 and good pharmacokinetic and pharmacodynamic properties have been reported to exert anti-inflammatory effects as an inhibitor of PTGS2 and NOS227. COX2 is the inducible form of cyclooxygenase and is responsible for the rate-limiting step in the conversion of arachidonic acid to prostanoids . Prostanoids have been well-characterized as proinflammatory mediators28. Both CASP3 and BCL2 were apoptosis-related proteins, which inhibited tumor growth by regulating and inducing cancer cell apoptosis29. C-T network analysis showed that naringenin and luteolin could target these above-mentioned targets.Of the 184 potential target nodes, many potential targets were linked with multiple compounds of different herbs, which may exhibit synergism effects or additive effects of PRGRC on gastric cancer. For example, NOS2 is associated with regulation of immune effector processTherefore, these compounds and targets might be closely related to gastric cancer, which demonstrated the potential therapeutic effect of PRGRC on gastric cancer through modulating the reaction of multiple compounds and multiple targets.31.Through integrating the KEGG and immune-related GO terms, we discovered 23 dramatically enriched pathways  that might be the main pathways and play a key role in gastric cancer. As shown in Fig. In summary, PRGRC played a role in the treatment of gastric cancer by acting on targets involved in cancer-related pathways.All of the disorders have common features or conjoint cellular processes, like the activation of a stress signaling pathway and the concomitant production of inflammatory cytokines. Hence, to further elucidate the mechanism of PRGRC, we collected the targets and pathways closely related to gastric cancer. Then, we assembled the “gastric cancer pathway” according to the pathological and clinical data. As shown in Fig. 32 and TNF-α33, and the activation of NF-κB caused inflammation of the gastrointestinal mucosa, leading to the occurrence of gastric cancer34. IL-2 was proved to be a T-cell growth factor that enhanced T cells and NK cell-mediated immune responses35. Besides, The literatures revealed that IL-10 acted as a multifunctional negative regulator participating in the bioregulation of immune cells, inflammatory cells and other cells36. As shown in Fig. Helicobacter pylori37. All these indicated that PRGRC might treat gastric cancer through the regulation of immune and inflammatory responses.Humoral and cell-mediated immune mechanisms were likely to be involved in the treatment of gastric cancer. As shown in Fig. 38. Abnormal apoptosis and excessive proliferation of gastric epithelial cells induced to the occurrence of gastric mucosal lesions, which in turn caused gastric cancer39. The literatures indicated that gastric cancer was closely related to Helicobacter pylori infection which increased the apoptosis of gastric epithelial cells40. Cytotoxin-associated gene A (CagA) and Vacuolating cytotoxin gene A (VacA) were the main pathogenic factors of Helicobacter pylori39, which could induce the production of various cytokines in the gastric mucosa, leading to apoptosis of mucosal epithelial cells42. It has been reported that aloe-emodin (MOL25) and emodin (MOL33) could inhibit the growth of Helicobacter pylori and reduce the apoptosis of gastric mucosa cells caused by CagA and VacA, thereby protecting the gastric mucosa and achieving the purpose of treating gastric cancer43.Apoptosis is a process of programmed cell death that occurs in multicellular organisms. Under normal circumstances, homeostasis is maintained by the balance between cell proliferation and apoptosis45. Caspase-3, a key pro-apoptotic factor, could be activated as cleaved-CASP3 to regulate the abnormal apoptosis of gastric mucosal epithelial cells46. Studies have found that BCL2 could not only act as an upstream factor of CASP3, but also as a direct substrate of CASP3 in the downstream, both of which were interrelated and mutually restricted in the process of apoptosis transmission48.Previous studies have confirmed that bcl-2 family played an important role in the apoptosis of gastric mucosa. When gastric cancer occurred, BCL2 inhibited the abnormal apoptosis of gastric mucosal cells by targeting pro-apoptotic proteins to the mitochondrial membrane, thereby preventing the development of gastric cancer49.PARP (poly (ADP-ribose) polymerase) family proteins were essential in the repair of single-stranded DNA breaks via the base excision repair pathway. PARP1 was a highly conserved DNA-binding protein and was the most ubiquitously expressed member of the PARP family. Numerous studies suggested that PARP1 was involved in maintaining genomic stability as well as regulating DNA repair and transcriptional processes. PARP1 was now considered to be a central regulatory hub of cell survival and cell death as well as a key component of a number of transcription factors involved in tumor development and inflammation. CASP3 could cleave PARP1 with full length of 116 kDa into two fragments of 31 kDa and 85 kDa, resulting in PARP1 not functioning properly and causing DNA breakage. The degradation of PARP1 was one of the hallmarks of apoptosisNaringenin (MOL71) and luteolin (MOL75) regulated the expression of BCL2 and CASP3 and indirectly regulated the expression of PAPR1 to inhibit the development of gastric cancer. Thus, all above suggested that the active ingredients in PRGRC were able to activate various cytokines and pathways and might treat gastric cancer by regulating immunity and apoptosis.From the pathways analysis, the synergistic effects of active compounds on gastric cancer were identified. Naringenin (MOL71)  and luteolin (MOL75) , screened via systems pharmacology approach, have been identified to exhibit good pharmacodynamic properties. Thus, we selected naringenin and luteolin as representative compounds to design the in vitro experiments to investigate the efficacy on cancer cells and inflammatory cells.50 of RAW264.7 cells treated with naringenin and luteolin were 140.9 μM and 27.33 μM, respectively  and luteolin  were selected for the follow-up experiments.CCK-8 assay showed that the ICTo further assess the potential anti-inflammatory effects of naringenin and luteolin, we used RAW264.7 cells treated by LPS with or without naringenin and luteolin in vitro. Thereafter, we verified the protein expression levels of PTGS2, NOS2 or RELA by western blot to confirm whether these two compounds have anti-inflammatory effects. The results in Fig. Meanwhile, we performed western blot on BCL2, CASP3, cleaved-CASP3 and cleaved-PARP1 proteins in SGC-7901 and AGS cells to evaluate the anti-apoptotic effect of naringenin and luteolin. The results for SGC-7901 cells were shown in Fig. Taken together, in vitro experiments indicated the molecular mechanisms of PRGRC on gastric cancer including regulation of immunity and promotion of apoptosis, which further validated the results obtained through systems pharmacology and ensured the reliability of in silico screen strategy.Gastric cancer has become one of the most common cancers due to high mortality and morbidity. Clinically, adverse drug reaction, drug resistance and cancer recurrence frequently occur, causing poor prognosis in gastric cancer patients. Therefore, the search for safe drugs with low side effects is necessary and urgent.In this study, we selected PRGRC commonly used in clinical practice as an example, and proposed the systems pharmacology approach to interpret the synergistic pharmacological mechanism of the TCM formula in the treatment of gastric cancer. Firstly, based on the evaluation methods, 83 active ingredients were obtained, and 184 potential disease-related targets were predicted. The results indicated that PRGRC has the features of multiple compounds and multiple targets. Both active compounds and C-T analysis then attested that several active compounds in PRGRC were imperative for the treatment of gastric cancer, including naringenin and luteolin. In addition, some targets such as PTGS2, NOS2, CASP3, and BCL2 have been confirmed to have anti-inflammatory and pro-apoptotic effects on gastric cancer cells. Moreover, the results of pathway and GOBP enrichment analysis, T-P analysis and integrated the "gastric cancer pathway" suggested that PRGRC primarily treated gastric cancer by modulating inflammatory response and apoptosis. Finally, in vitro cytotoxicity test showed that naringenin and luteolin had obvious antiproliferative effect on gastric cancer cells. Besides, we verified that naringenin and luteolin has significant anti-inflammatory effect and regulate apoptosis of gastric cancer cells by western blot and flow cytometry.In summary, the systems pharmacology approach revealed the complex molecular mechanisms of PRGRC for gastric cancer and elucidated the multi-molecular and multi-target synergistic effects of TCM. Moreover, this strategy provided a potential approach for rational discovery of new drugs. In the development of new drugs, we can try to determine the pharmacological activity of compounds by predicting their relevant targets and diseases. This approach accelerated the process of new drug development and promoted the modernization of TCM.50 .The compounds contained in PRGRC were collected from the Traditional Chinese Medicine Systems Pharmacology Database which has been developed by our previous studies51. And the thresholds for OB and DL were respectively set to 30% and 0.18.PRGRC is an oral formulation and must undergo the ADME process to reach the target organs and tissues to exert their efficacy. In this article, we employed OB and DL properties as parameters for screening bioactive compoundsTargets are usually bioactive macromolecules in the body that recognize and bind pharmacodynamic molecules for the purpose of treating disease. Hence, target prediction plays an important role in elucidating the molecular mechanisms and drug actions associated with gastric cancer. In this work, we predicted the direct and indirect efficacy targets of PRGRC through the WES and SysDT algorithms, respectively.14.The WES algorithm used chemical molecular fingerprints and Dragon parameters to calculate the weighted Tanimoto similarity between compounds and protein ligands, and then described the Global Similarity of compounds and protein ligands by pooled similarity. As a novel tool, the obtained model performed well in predicting the binding with average sensitivity of 85% (SEN) and the non-binding patterns with 71% (SPE) with the average areas under the receiver operating curves  of 85.2% and an average concordance of 77.5%in-house computer model based on two powerful mathematical tools of Random Forest (RF) and Support Vector Machine (SVM) to model drugs and targets and their interactions by integrating information from large-scale chemical, genomic and drug data. The model had an overall accuracy of 97.3%, 87.7% accuracy in activation prediction and 99.8% accuracy in inhibition prediction. To capture more potential components, this study defined the filtering criteria as RF value ≥ 0.7 or SVM ≥ 0.815.SysDT is an http://ctdbase.org/) for disease enrichment analysis to obtain the potential targets associated with gastric cancer.We mapped the targets predicted above to the CTD database 53 and DAVID (http://david.abcc.ncifcrf.gov) database54. Only P-value < 0.01 were selected.In order to further probe the involved pathways and the biological processes of the targets obtained above, we mapped the targets to the KEGG . The samples were dissolved in DMSO to obtain a stock solution at a concentration of 100 mM and then stored at 4 °C. The dilution volume of DMSO finally added to the medium did not exceed 1% of the total volume, to ensure that DMSO has no effect on cell viability.2 at 37 °C.Mouse macrophage cells RAW264.7, human gastric cancer cells SGC7901 and AGS were purchased from Chinese Academy of Sciences Shanghai cell bank. RAW264.7 cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) with 10% foetal bovine serum (FBS) (Gibco BRL). SGC7901 and AGS cells were cultured in RPMI-1640 medium with 10% FBS. All experiments cells were maintained in a humidified atmosphere of 5% CO4 cells/ml for 24 h, and then cells were treated with or without various indicated concentrations of naringenin or luteolin, and then cells viability were measured by CCK-8 after 24 h. The various indicated concentrations of naringenin or luteolin were respectively 10, 50 and 100 μM or 5, 10 and 15 μM, respectively.Cell viability was estimated by CCK-8 assay (BestBio). RAW264.7, SGC-7901 and AGS cells were seeded into 96-well plate at a density of 8 × 106) were cultured in 100 mm diameter dishes for 24 h and then treated with or without various indicated concentrations of naringenin or luteolin for 2 h before treated with 1 μg/ml LPS ), after that, cells were cultured in a humidified incubator with 5% CO2/95% O2 at 37 °C for 18 h. The cells were collected at the end of the culture for western blot, which were used as a detection of inflammatory mediators.RAW264.7 cells  overnight. Blots were then incubated in secondary antibody for 1 h before being developed with ECL chemiluminescence detection kit (Bio-Rad Laboratories). The images of the original blots were obtained by Image Lab software.5 cells/ml. Cells were stained with annexin V-FITC for 15 min and propidium (PI) for 5 min in the dark before analysis by a flow cytometer .Cell apoptosis was determined using a FITC Annexin V apoptosis kit (BD Bioscience) according to the manufacturer’s instructions. Following treatment with various concentrations of naringenin and luteolin for 24 h, the cell suspension was prepared using trypsin and centrifuged at 1000 rpm for 3 min then rinsed with ice-cold PBS. Cells were then resuspended in binding buffer at a concentration of 1 × 10Statistical analysis were performed GraphPad Prism 8.0 (Graph Pad Software Inc). All data were presented as means ± standard error and repeated at least two or three independent experiments with the same result.Supplementary Figures.Supplementary Tables.