Fasciola hepatica) was collated by members of the European COST Action “COMBAR” (Combatting Anthelmintic Resistance in Ruminants), and combined with data from a previous systematic review of AR in GIN. A total of 197 publications on AR in GIN were available for analysis, representing 535 studies in 22 countries and spanning the period 1980–2020. Reports of AR were present throughout the European continent and some reports indicated high within-country prevalence. Heuristic sample size-weighted estimates of European AR prevalence over the whole study period, stratified by anthelmintic class, varied between 0 and 48%. Estimated regional (country) prevalence was highly heterogeneous, ranging between 0% and 100% depending on livestock sector and anthelmintic class, and generally increased with increasing research effort in a country. In the few countries with adequate longitudinal data, there was a tendency towards increasing AR over time for all anthelmintic classes in GIN: aggregated results in sheep and goats since 2010 reveal an average prevalence of resistance to benzimidazoles (BZ) of 86%, macrocyclic lactones except moxidectin (ML) 52%, levamisole (LEV) 48%, and moxidectin (MOX) 21%. All major GIN genera survived treatment in various studies. In cattle, prevalence of AR varied between anthelmintic classes from 0–100% (BZ and ML), 0–17% (LEV) and 0–73% (MOX), and both Cooperia and Ostertagia survived treatment. Suspected AR in F. hepatica was reported in 21 studies spanning 6 countries. For GIN and particularly F. hepatica, there was a bias towards preferential sampling of individual farms with suspected AR, and research effort was biased towards Western Europe and particularly the United Kingdom. Ongoing capture of future results in the live database, efforts to avoid bias in farm recruitment, more accurate tests for AR, and stronger appreciation of the importance of AR among the agricultural industry and policy makers, will support more sophisticated analyses of factors contributing to AR and effective strategies to slow its spread.Helminth infections are ubiquitous in grazing ruminant production systems, and are responsible for significant costs and production losses. Anthelmintic Resistance (AR) in parasites is now widespread throughout Europe, although there are still gaps in our knowledge in some regions and countries. AR is a major threat to the sustainability of modern ruminant livestock production, resulting in reduced productivity, compromised animal health and welfare, and increased greenhouse gas emissions through increased parasitism and farm inputs. A better understanding of the extent of AR in Europe is needed to develop and advocate more sustainable parasite control approaches. A database of European published and unpublished AR research on gastrointestinal nematodes (GIN) and liver fluke ( Fasciola hepatica) threatens the profitability and sustainability of livestock production. Helminths affect growth [Livestock production is estimated to contribute on average 40% of agricultural production value worldwide  . The inft growth , productt growth  and reprt growth , and thrt growth . Furthert growth  and GIN t growth , 29, 47.F. hepatica) may also be widespread , levamisoles (LEV) and macrocyclic lactones  to maintain infections below levels that can cause clinical and sub-clinical disease. However, Anthelmintic Resistance (AR) in GIN has been reported worldwide in multiple nematode species, against most anthelmintic classes and sometimes simultaneously multiple different classes , 42, 49 ad e.g. , 18, 25), 25F. hehttps://www.scops.org.uk) and Control of Worms Sustainably (https://www.cattleparasites.org.uk), yet farmers continue to implement AR-selective practices, such as moving sheep to low challenge or “clean” pastures immediately after treatment and treating ewes and lambs more frequently than necessary [Newer, more sustainable, approaches to helminth control, which reduce the frequency of anthelmintic treatments  while maintaining high productivity levels are available. These approaches include Targeted Selective Treatment (TST), based on individual animal health or production parameters , 34, or ecessary . Confirmecessary . Similarecessary . This suhttps://www.combar-ca.eu) established a consortium of researchers from 26 countries across Europe. The consortium aims to advance research on the prevention of AR in helminth parasites of ruminants and disseminate current knowledge among all relevant stakeholders through improving diagnosis, understanding the socio-economic aspects of infection and decision making, and developing innovative, sustainable control methods. As part of this, and to encourage the uptake of COMBAR outputs and recommendations for sustainable control of helminth parasites, an up-to-date synthesis of current AR research is needed.A systematic review of reports of AR in European ruminant livestock confirmed that AR is spatially widespread, but also highlighted limitations in the available data, due to sample selection bias, a lack of georeferenced data to allow robust spatial analyses, and a number of countries for which no data existed . In 2017In this study, we draw on the collective expertise and data held by the COMBAR consortium to address the limitations of AR research identified previously, by collating published and unpublished data on AR in Europe that can be used to support advocacy for sustainable control programmes and to target future research efforts where they are needed most.This review was performed and is reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) : (1) preA standardised spreadsheet model was populated with data from the previous systematic review . MembersEach publication or unpublished dataset, hereafter referred to as “publication”, was given a unique identifying number. Within each publication, multiple anthelmintic classes and livestock classes tested were entered as separate entries, hereafter referred to as “studies”. Therefore, one “publication” may contain several “studies”. When a publication presented separate data for different regions within the same country, these data were split into separate rows (studies) in the database (e.g. ). When aFor GIN, data were stratified primarily by host species and anthelmintic class, with the exception of the ML, which were divided into avermectins (e.g. ivermectin) and milbemycins . Hereafter, MOX is analysed separately to the other ML. Other classes were benzimidazoles (BZ), imidazothiazoles, i.e. levamisole (LEV), amino-acetonitrile derivatives, i.e. monepantel (MPTL), and salicylanilides, i.e. closantel (CLOS). Studies testing more than one anthelmintic simultaneously in the same animals were entered as combination treatments (COMB), except where the combination involved MOX and ivermectin; in this case the anthelmintic class was entered as MOX due to its relatively high efficacy against some ivermectin-resistant populations of GIN, and observations that the extent of cross-resistance is greater in ivermectin than MOX-resistant populations (reviewed by ).F. hepatica, data were stratified by anthelmintic compound, rather than class, in order to differentiate between the activity of triclabendazole (TBZ) and albendazole (ALB), which have different efficacy profiles depending on the age of the fluke [F. hepatica are genetically similar between host species [For he fluke . Data fo species .in vivo; Faecal Egg Count Reduction Test (FECRT) and Controlled Efficacy Test (CET), in vitro, and molecular methods) is unavailable, therefore increasing the timeliness of the analysis and potentially reducing limitations such as regional publication bias. Some misclassification of resistance is consequently expected in both directions, but the increased number of studies adds power to analysis of trends.Due to significant upcoming changes in protocols for detecting AR , inclusiData analysis was completed in R v3.6.1 . Figuresx = the proportion of farms with AR, w = weights :GIN AR prevalence for each anthelmintic class and ruminant species were estimated from the reported prevalences and were weighted by sample size (number of farms in each study) to give a weighted mean (I2 statistic [−7) to allow variance to be calculated as input for Cochran’s Q statistic when estimated prevalence was zero. Negative I2 values were set to zero [I2 values were not estimated where only one study existed. Higher I2 values indicate higher levels of heterogeneity.Heterogeneity in AR prevalence estimates between studies (aggregated by anthelmintic class and host species) was estimated using the tatistic , calculatatistic , with th to zero , as wellI2 were also estimated using only studies investigating 10 or more farms.To evaluate the impact of sample selection bias in studies with small sample sizes , sample size-weighted mean prevalences, standard deviations and F. hepatica due to insufficient studies.Similar analyses were not completed for ” function in R) and estimates for the cost of helminth infections and research budget for projects starting after 01/01/2008 and ending before 31/12/2017 [Qualitative assessment of spatial bias in AR research was made by mapping the number of publications per ruminant species per country. Quantitative assessment of spatial bias in AR research (GIN only) was made by relating the number of studies per country and the research budget or cost of helminth infection per country using Spearman rank correlation  for each active compound, and na is the number of actives to which resistance was documented to date. For GIN, prevalence for up to four actives was included: BZ, LEV, ML, and MOX separately from other MLs as a special case (as detailed above). MPTL was not included in the prevalence part of the equation as it has been tested only rarely to date, and the same applies for the narrow-spectrum active sulphonamide, CLOS. Both, however, were added to na in countries in which cases of resistance had been confirmed. Thus, for instance, a country with reports of AR involving BZ and ML with sample-size weighted prevalence of 0.5 and 0.1, would score (0.5 + 0.1 + 2 = 2.6), while another reporting resistance to BZ, LEV, ML, MOX and MPTL with prevalence, respectively of 1, 0.7, 0.8, 0.4 and 0.1, would score (1 + 0.7 + 0.8 + 0.4 + 5 = 7.9). For liver fluke, seven actives were included in both p and na (see Results).To compare the estimated level of AR between countries alongside the relative confidence that can be placed in that estimate, an index was compiled for each, as follows:S is the number of separate publications (or unpublished reports) in the database, F the total number of farms included, and A the total number of anthelmintic classes investigated. F is the sum of the total number of farms used per study, regardless of how many anthelmintics were assessed on each, to prevent double counting. Both S and F were log10-transformed to stabilise variance, and scaled to the maximum value across countries , to provide a measure of the relative research effort among countries in the database at the time of the analysis. One publication from Ireland included data from 4211 farms but used pooled faecal samples collected by farmers: for this case, the number of farms was divided by an arbitrary value of 10 before entry into the equation, to avoid unfair comparison with studies using full FECRT. The confidence index itself is arbitrary, but standardised across countries, and aims to assess the relative extent to which AR has been thoroughly investigated, to aid interpretation and prioritisation of future research.Confidence was not estimated from prevalence; rather, it is an index of research effort, taking into account the depth and breadth of investigation into the AR situation nationally:Level versus Confidence (= Research effort) were plotted for each country: separately for GIN in either sheep, goats or cattle, and for fluke for all host species combined. Data manipulation and analysis were completed using Excel .Results of Anthelmintic Resistance is a dynamic process, evolving as helminth populations experience repeated treatment, and as farmers change to more effective anthelmintics when treatment failure is recognised. Research on AR is also dynamic, often responding initially to reports of treatment failure on particular farms, before expanding to prospective surveys, with highly variable selection criteria, and assessing additional anthelmintic groups as they become available or fall under suspicion of failure. Because of this, prevalence data averaged over a long period are unlikely to accurately capture the current situation in any given country, or trends in AR over time.To address these confounders, change in prevalence was evaluated over time. Reports of AR on single farms were excluded, since these are often conducted in response to suspected treatment failure. Prevalence was transformed to arcsine of the square root of prevalence in order to stabilise the variance around values close to 0 or 1. The relationship between year and prevalence was then tested using Pearson and Spearman rank correlation.Nematodirus battus was also excluded. Initially, average prevalence was plotted for each anthelmintic group for each decade, to visualise trends in AR over time. The database was then searched separately for each anthelmintic group in GIN in sheep, goats and cattle, and fluke in all hosts. For each country meeting these criteria , correlation between prevalence and year was assessed as above. Where a significant correlation was confirmed, linear regression was conducted to evaluate the rate of change in AR prevalence over time.In a second stage analysis, the data set was reduced to countries that reported at least three studies in different years, to further decrease the influence of responsive investigations and to capture change over time within countries. Sample-size adjusted prevalence, while arguably giving the fairest indication of how common AR is among farms in a given sample set, can result in bias: for example, if larger surveys are conducted as concern over resistance rises over time, more recent data will dominate overall prevalence estimates. For this reason, prevalence from individual studies was averaged over each decade without adjusting for sample size. Studies in sheep and goats were combined to increase power, given that helminth species are largely shared between these hosts. Italy was excluded because its combination of low AR prevalence and high level of reporting in recent years (19/20 reports in sheep and goats coming after 2007) strongly affected the overall trajectory across countries, and a single large study in the UK focusing only on The main relevant influences that might affect research on AR were identified by SWOT  analysis, to inform further AR research. The SWOT template was drafted by the lead authors and circulated for input by all co-authors.Supplementary Material. A live version of the database is also freely available on the Open Science Framework [https://www.gnu.org/licenses/gpl-3.0.html).The database used in these analyses is provided as ramework  and submramework . This vehttps://www.parasite-journal.org/10.1051/parasite/2020062">Fig. S1). The majority of studies investigated AR in GIN on single farms . 23.4% (n = 47) of the “publications” were unpublished.In all, 197 publications were available for analysis, representing 535 studies, 22 countries and spanning the period 1980–2020 (p = 0.49) but was positively correlated with the economic cost of helminth infections estimated in the same study .There was a regional research bias towards Western Europe and especially the UK, while Eastern European countries were relatively underrepresented in the body of AR research . AR was most widely reported in sheep, whereas AR in cattle and goats was understudied in many regions, indicated by the number of countries with no data available .Reports of AR were widespread throughout Europe, particularly to BZ . However, there was considerable heterogeneity between studies . TBZ resistance was reported in the UK, Ireland, Spain and the Netherlands. ALB resistance was reported in Sweden and Spain. CLOS resistance was reported in Sweden, and clorsulon resistance was reported in Spain. No resistance was observed in Italy. The majority of publications investigated AR on individual farms, with the exception of two larger studies. Kamaludeen et al. [F. hepatica originated from the Netherlands and were reported on 94 farms by the Royal Animal Health Service between 1998 and 2003 (https://www.gddiergezondheid.nl).Anthelmintic Resistance in n et al.  identifin et al.  identifiρ18 = 0.75, p = 0.001; and ρ15 = 0.79, p < 0.001, respectively), but this relationship was not apparent for studies of GIN in cattle (p = 0.46), or for fluke across host species .For GIN in sheep and goats, the observed level of treatment failure correlated with research effort  and in the UK for BZ  and ML . Correlation in other countries was mostly positive but not significant. Linear regression of prevalence against year ) produced similar slopes for the rate of increase in BZ in Switzerland and the UK, equating to approximately 20% increase in prevalence per decade, and a faster rate of increase of 33% per decade for ML.For GIN in sheep, six countries reported at least three studies in different years, excluding single-farm case reports: France (BZ), Italy , the Netherlands , Slovakia (BZ) Switzerland (BZ), and the United Kingdom . No countries met this criterion for GIN in goats or cattle, or fluke. Further analysis of these 11 country-anthelmintic group combinations for GIN in sheep, on arcsine-transformed prevalence and with a Bonferroni-adjusted critical The main relevant influences that might affect research on AR that were identified by SWOT analysis . Prevalence of AR in GIN against BZ, LEV, ML and MOX ranged between 0% and 48% when averaged over the entire database, but confidence intervals were wide and there was a high level of heterogeneity between studies. There was also considerable variation within and between regions depending on host species and anthelmintic class. Ten studies were largely in agreement that AR against MPTL in sheep is currently rare in Europe, although these studies were focussed on only six countries. AR in https://www.parasite-journal.org/10.1051/parasite/2020062">Fig. S4) is reflected in the spatial distribution of publications per country , and implementing looser inclusion criteria, allowed the inclusion of data from more countries than in previous reviews , publica country . FailingThe relationship between research effort and observed AR was investigated semi-quantitatively using composite indices of level of AR across anthelmintic groups and research effort or confidence, per country. Level of AR included the number of anthelmintic groups to which AR was reported and observed prevalence of resistance to each (i.e. proportion of farms with AR). Research effort, indicative of relative confidence in the levels of AR, comprised the number of studies in the database, the total number of farms investigated, and the number of anthelmintic groups tested. The positive relationship between level of AR and research effort in the better-studied systems  is to be expected for two main reasons. Firstly, as more studies are conducted and more anthelmintic groups tested, the chance of finding AR increases. Secondly, a perceived problem with anthelmintic efficacy is often the stimulus for investigations, and finding AR will provide justification for further and larger studies, fuelling a cycle of perceived and observed AR. To some extent, therefore, differences between countries in both observed level of treatment failure and research effort represent different positions on a trajectory of learning about existing AR problems, as well as between-country heterogeneity in true levels of AR. Nevertheless, the fact that low research effort is invariably associated with low apparent levels of treatment failure should stimulate further studies, especially in those countries positioned towards the lower left in Overall confidence in AR status in GIN in cattle, and in fluke in both sheep and cattle, was very low, and further studies should also be prioritised to address these knowledge gaps in all countries. In sheep and goats, high confidence can be placed in high levels of AR in several countries  and limited time range, as well as potential bias in farm selection. However, with the notable exception of LEV, for which some surveys gave inaccurate results before the mid-90s , successIncreasing AR is less obvious after 2009 for LEV, although sample sizes are low and there is no robust evidence of a decrease in prevalence. Studies in New Zealand have suggested that a change to less intensive treatment regimens might be successful in slowing or even reversing anthelmintic resistance in GIN populations . The relThe analyses of AR revealed several sources of bias, which can confound interpretation and impede solutions-oriented research. Thus, bias makes it very difficult to identify risk factors for rapid development of AR, and to determine whether differences in observed levels of treatment failure between countries, and between farms, can be used to guide future strategies. While removal of bias in studies of AR is difficult and perhaps impossible, greater efforts to reduce or account for it could support more refined analyses and better understanding of regional patterns and risk factors on which to base advice to farmers and policy makers.SWOT analysis also identified potential biases, and within-country variation that could affect current and future AR research. Although data protection legislation  may limit the resolution of location data that can be published, there is now an opportunity to improve reporting of AR research to facilitate future geostatistical analyses. Future studies should strive to publish the location of farms in a way that protects farmer anonymity e.g. publishing latitudes and longitudes to one decimal place would give an indicative spatial accuracy of ±10 km at the equator.https://cordis.europa.eu/project/id/22851) GLOWORM , DELIVER  and PARAGONE . As a result, AR is now considered part of the broader antimicrobial resistance problem and stakeholders have recently recognised the emerging risk and agreed that it is now time to take concrete actions to ensure the responsible use of all veterinary medicines, including anthelmintics in livestock species [During past decades, European research and investment tended to focus on resistance to antibiotics, but AR research effort accelerated after the turn of the millennium, coinciding with investment by the European Commission in several AR research consortia: PARASOL  was lower than on non-randomly selected farms . Similarly, small sample sizes  may inflate uncertainty. Study design includes the active compounds to be tested and whether or not to include an untreated control group, which are also subject to farmer consent: FECRT with all five licensed groups for sheep (including an avermectin and moxidectin) and a control group, with 15 animals in each, would require 105 animals, which is often unrealistic on commercial farms due to the time and cost of sampling; consequently, most studies test only a limited range of anthelmintics. The conditions for inclusion of animals in the test, such as pre-treatment Faecal Egg Count (FEC) varies between studies, as does the detection limit of the FEC method used, and the extent of species identification. The gold standard for assessing AR, the CET, requires slaughter of both control animals and treated animals, and is not suitable for monitoring on commercial farms. Each FECRT or in vitro method is open to error, e.g. FECs are an indirect measure of anthelmintic efficacy against adult helminths. Sample size within the existing database was not sufficiently high to compare AR prevalence across countries while taking account of variation in method and would be further undermined by concealed variation and bias.Non-random sampling, for example, due to researchers selecting farms with suspected AR, or farmers self-selecting to participate (possibly because they suspect they have AR on their farm) may inflate estimated prevalence. This is supported by a survey of both randomly selected and non-randomly selected sheep flocks in Norway  where thFrequent anthelmintic treatment is held as one of the major reasons for the genesis of resistant GIN populations, and a number of approaches are advocated to slow the development of AR . ExtensiIn Switzerland, a programme was established in 1999 by the Small Ruminant Health Service to provide an annual service for sheep and goat farmers based on quantitative faecal examination. The programme, now including about 1400 farms, is partially subsidized by the government, covering about one third of the costs. Pooled samples are analysed from different groups of animals and, based on the results, veterinary advice is given with respect to anthelmintic treatment and further sampling. An evaluation performed five years after establishment of the program revealed that, on 73% of the farms, GIN were controlled successfully, with a mean annual treatment frequency of 2.0 compared with 3.4 on farms that did not participate in the programme. Participation in faecal monitoring also strengthened collaboration with the veterinarian – according to the farmers, the support of the veterinarian was “good” or “very good” in 86% of cases (unpublished data).In Italy, the Regional Centre for Monitoring Parasitic Infections  has been established since 2000 by the Department of Agriculture and Livestock Production of the Campania region and is economically supported by the farmers’ associations of Campania and other Italian regions. The strategies for the management of livestock infections caused by GIN, liver flukes and other parasites are based on promoting: best practice for diagnosis, regular monitoring on-farm (at least three FEC per year), best practice for treatment, monitoring the efficacy of treatments through the FECRT, and tailored advice to farm veterinarians on sustainable parasite control. These recommendations and activities are now well integrated into routine farm management of large ruminants  and small ruminants (sheep and goats). The programme includes over 1000 farms per year. A survey performed for the last decade revealed that, on 88% of the farms, GIN were controlled successfully, with a lower mean annual treatment frequency of 2.0 compared with 3.1 on farms that did not participate in the programme. Participation in faecal monitoring also strengthened collaboration with the veterinarian – according to the farmers the support of veterinarian was “very good” in 94% of cases.Recent studies suggest that improving awareness of helminth control and AR may improve the uptake of sustainable parasite control practices . Jack etF. hepatica are increasing, confirming AR presence on small numbers of farms may no longer be necessary. The introduction of more rigorous methods for detecting AR is an opportunity to shift focus to employing robust randomised sampling methods that would allow more detailed epidemiological investigations, including improved estimates of regional prevalence and risk factor analysis.AR is present across the European continent and despite the limitations of the current body of AR research, the analyses presented here provide useful indicators of the potential scale and potential future trajectory of the problem of AR in Europe, and may help improve the uptake of sustainable control practices. As the presence of AR in GIN is now widespread globally, and reports of AR in The authors have no conflicts of interest to declare.supplementary data files.LR, ERM and HRV conceived the project. HRV and LR collated data. HRV, ERM and LR conducted analyses and drafted the manuscript. All authors developed the database by contributing data and participating in quality control, assisted with technical editing of the manuscript, and approved the final manuscript and https://www.parasite-journal.org/10.1051/parasite/2020062The supplementary material of this article is available at Figure S1. Number of publications (peer-reviewed papers or theses) investigating AR in GIN in ruminants.Figure S2. Estimated prevalence of resistance against the benzimidazoles (BZ), levamisole (LEV), avermectins , and moxidectin (MOX) in gastrointestinal nematodes in goats.Figure S3. Estimated prevalence of resistance against the benzimidazoles (BZ), levamisole (LEV), avermectins , and moxidectin (MOX) in gastrointestinal nematodes in cattle.Figure S4. Countries reporting at least one case of anthelmintic resistance in gastrointestinal nematodes infecting sheep, goats and cattle in Europe.Table S1. Mean (SD in brackets) sample size weighted prevalence of anthelmintic resistance in sheep stratified by country and anthelmintic class.Table S2. Mean (SD in brackets) sample size weighted prevalence of anthelmintic resistance in cattle stratified by country and anthelmintic class.Table S3. Mean (SD in brackets) sample size weighted prevalence of anthelmintic resistance in goats stratified by country and anthelmintic class.Table S4. Regression analysis results for prevalence of anthelmintic failure over time.Table S4a. Benzimidazole efficacy in sheep in Switzerland.Table S4b. Benzimidazole efficacy in sheep in the UK.Table S4c. Ivermectin efficacy in sheep in the UK.


The high mobility group box 1 (HMGB1) is a well-known late mediator of sepsis, secreted by multiple stimuli, involving pathways, such as the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways, and reactive oxygen species (ROS) under inflammation. Sulfatide, in contrast, is a sphingolipid commonly found in myelin sheets with a disputed immunological role. We sought to determine the immunological characteristics of sulfatide in the periphery by analyzing the secretion of HMGB1 triggered by lipopolysaccharide (LPS) stimulation in Raw 264.7 cells. Suppression of HMGB1 secretion by inhibiting its cytosolic translocation was observed after pre-treatment with sulfatide before LPS stimulation. Further analysis of the downstream molecules of toll-like receptor (TLR) signaling revealed suppression of c-Jun N-terminal kinase (JNK) phosphorylation and p65 translocation. LPS-mediated ROS production was also decreased when sulfatide pre-treatment was provided, caused by the down-regulation of the phosphorylation of activators, such as IRAK4 and TBK1. Investigation of the upstream mechanism that encompasses all the aforementioned inhibitory characteristics unveiled the involvement of lipid rafts. In addition to the co-localization of biotinylated sulfatide and monosialotetrahexosylganglioside, a decrease in LPS-induced co-localization of TLR4 and lipid raft markers was observed when sulfatide treatment was given before LPS stimulation. Overall, sulfatide was found to exert its anti-inflammatory properties by hindering the co-localization of TLR4 and lipid rafts, nullifying the effect of LPS on TLR4 signaling. Similar effects of sulfatide were also confirmed in the LPS-mediated murine experimental sepsis model, showing decreased levels of serum HMGB1, increased survivability, and reduced pathological severity. Approximately 45 years have passed since the HMGB1 protein, an abundant nuclear protein and a well-defined danger-associated molecular pattern (DAMP) molecule, was first purified  and DNA HMGB1 can be either passively released through non-apoptotic cell death, such as in necrotic cells , or actiMost studies addressing the immunological role of HMGB1 have focused on the role of extracellular HMGB1 as a DAMP molecule and its chemokine-like behavior. Depending on its redox status, HMGB1 exerts different characteristics: 1) as a thiol isoform, in which all of the three active cysteine residues  are in free-thiol(-SH) form, HMGB1 binds to C-X-C motif ligand 12 (CXCL12) and shows chemokine-like activity, recruiting immune cells to the site of inflammation ; 2) the Sulfatide, also known as 3-O-sulfogalactosylceramide, is a lipid commonly found in the myelin sheath in both the central and peripheral nervous system . First iIn this study, we aimed to elucidate the effect of sulfatide in the context of innate immunity by investigating its effect on HMGB1 secretion under LPS stimulus and discuss the specific molecules involved in the process.Raw 264.7 cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% heat-inactivated fetal bovine serum , 100 μg/mL of penicillin, and 100 μg/mL of streptomycin . Treatment was performed after allowing the cells to adapt to Opti-MEM (Gibco) for 2 h, after which the media was replaced.Escherichia coli O111:B4; > 3 x 106 EU/mL; Sigma), sulfatide , 18:0(2R-OH) sulfogalactosylceramide , C24:0 mono-sulfogalactosylceramide , C24:0 mono-sulfogalactosylceramide , galactosylceramide , and ceramide  were used as indicated in the figures. All experiments were performed using vehicle as a negative control.LPS  were housed in a SPF-grade facility with controlled temperature, humidity, and light. For all experiments, 8-week old female mice with approximate body weight of 20 g were used. The animals were ethically sacrificed, and the femur and tibia were extracted. Bone marrow was collected via warm, serum-free DMEM lavage until no remaining bone marrow was visible. Bone marrow was collected and filtered through cell strainer with 40 μm pore  to remove any undesirable debris and washed with excessive media to further remove unfiltered debris. The resulting cells were plated to 100 mm cell culture-treated dish , and then differentiated using 20 ng/mL GM-CSF in complete medium for 7 days to yield BMDMs.Culture media after treatment were collected after 24 h to compare HMGB1 secretion between groups. Culture media were then centrifuged at 3500 × g for 5 min to remove any cell debris. The supernatant was collected for trichloroacetic acid (TCA)/acetone precipitation. Then, 10% by volume of ice-cold TCA was added to the samples and mixed by inverting. After incubating overnight at−20°C, the samples were thawed and centrifuged at 20000 × g for 90 min. Supernatants were then discarded. The remaining pellets were washed with−20°C acetone by vortexing vigorously and left overnight at−20°C. Samples were centrifuged at 20000 × g for 90 min, and the resulting supernatants were removed. The remaining pellets were then dried and boiled with 2X sample buffer.Cells were harvested by scraping using cold Dulbecco's phosphate buffered saline (PBS) after the indicated time periods; they were then collected by centrifuging at 3000 × g for 5 min. Supernatants were discarded, and radioimmunoprecipitation (RIPA) buffer was added before sonication. Lysed cells were centrifuged at 20000 × g for 10 min to remove any debris. The resulting whole cell lysates were collected, and protein concentration was quantified using the bicinchoninic acid (BCA) assay. The cell lysates were then prepared by heating to 65°C for 10 min after adding sample buffer to minimize the loss of phosphorylated protein to beta-elimination.SDS-PAGE was performed on samples prepared via the abovementioned methods, and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane for western blotting. Transferred membranes were blocked using 5% skimmed milk. Primary antibodies for HMGB1 , JNK , ERK1/2 , p38 , phospho-IκBα , phospho-IRAK4, phospho-TBK1 , caveolin 1 , TLR4 , and β-actin (Santa Cruz) were diluted in 5% skimmed milk solution and incubated overnight at 4°C. After extensive washing, the corresponding secondary antibody solutions were incubated for 1 h at room temperature (20~25°C). The membranes were then washed, and signals were detected using enhanced chemiluminescence substrate solution  and X-ray film . Membranes were stripped using stripping solution  for re-blotting, as necessary. Densitometry analysis was performed using Image J.Raw 264.7 cells were seeded in 4-well chambered glass slides coated with poly-L-lysine (Sigma). Treatment dosage for LPS was increased to 200 ng/mL to facilitate visualization via immunofluorescence, and sulfatide dosage was adjusted accordingly to maintain molar ratio. Treatment was performed for the duration indicated in Figure Legends. After treatment, cells were then fixed with 4% paraformaldehyde overnight in 4°C. On the subsequent day, the cells were washed with PBS and permeabilized with 1% Triton X-100 and blocked with bovine serum albumin (BSA). Primary antibodies anti-p65 (Santa Cruz) or anti-HMGB1 (Abcam) were diluted in BSA solution and left to incubate overnight at 4°C. After thorough washing, the respective secondary antibodies conjugated with Alexa Fluor 488  were diluted in BSA solution and incubated at 37°C for 45 min. Slides were then washed, dried, and mounted using mounting medium containing 4′,6-diamidino-2-phenylindole . Sealed slides were observed via FV1000 confocal microscopy (Olympus). Localization of sulfatide was determined by treating Raw 264.7 cells with biotin-sulfatide and staining them with streptavidin-Alexa Fluor 488 (Invitrogen). Localization of TLR4 was detected using mouse anti-TLR4 antibodies (Invitrogen).Raw 264.7 cells were pre-treated with either vehicle control or 20 μM of sulfatide and with vehicle control or 100 ng/mL of LPS for 1 h. The treatment medium was removed, and culture dishes were washed twice with warm culture medium. H2-DCFDA  was treated as per the manufacturer's instructions. Cells were viewed under a fluorescence microscope. For flow cytometric analysis of ROS levels, the cells were detached before H2-DCFDA treatment.Raw 264.7 cells were seeded in 4-well chambered glass slides coated with poly-L-lysine (Sigma). Treatment dosage for LPS was increased to 1 μg/mL to maximize lipid raft formation and facilitate visualization via immunofluorescence, and sulfatide dosage was adjusted accordingly to maintain molar ratio. Treatment was performed for the duration indicated in Figure Legends. After treatment, the cells were then washed once with 4°C complete growth medium. Washed cells were incubated in cholera toxin B-Alexa Fluor 549 (Invitrogen) staining solution, prepared in 4°C complete growth medium. Cells were washed with ice-cold PBS three times and fixed with ice-cold 4% paraformaldehyde for 15 min.Raw 264.7 cells were treated with reagents for 8 min as indicated in the legends, and cells were briefly washed three times with ice-cold PBS to halt the internalization of lipid rafts. Cells were then lysed using the ice-cold buffer provided by Caveolea/Rafts Isolation Kit (Merck) supplemented with Triton X-100. Lysates then underwent ultracentrifugation with OptiPrep™ density gradient, provided by the aforementioned kit, and nine fractions were collected. Collected fractions were then supplemented with 1% SDS to assist complete dissociation of the protein from the lipids. Treated samples were concentrated using TCA/Acetone and analyzed by immunoblotting.Wild-type C57BL/6 mice obtained from Orient Bio  were housed in a SPF-grade facility with controlled temperature, humidity, and light. For all experiments, 8-week old female mice were used. For serum collection, mice were anesthetized using an isoflurane–oxygen mixture, and combinations of PBS, LPS (3 mg/kg), or sulfatide (25 nmol) were injected with a total of 100 μL injection volume, intraperitoneally. The animals were allowed 60 min between injections to fully recover from the effects of anesthesia. Serum samples were collected after 18 h. Survival rate was measured by following the same procedure as mentioned above, with increased doses of LPS and sulfatide injection . Mice were checked twice every day and observed until completion. Survival data were then analyzed through Kaplan-Meier survival analysis. Pathological scores were obtained using the scoring regimen described by Shrum et al. , and theTumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) ELISA were performed using Raw 264.7 cell culture medium. Cells were treated with vehicle control (negative control), 100 ng/mL LPS, and sulfatide 20 μM, followed by LPS 100 ng/mL. Culture media were collected after 12 h of treatment and centrifuged to remove any cell debris. ELISA was performed with the resulting supernatant following the manufacturer's instructions (Invitrogen). Serum obtained from murine experimental sepsis models was analyzed for HMGB1 levels with a HMGB1 ELISA kit , following the manufacturer's instructions.post-hoc test, using GraphPad Prism 5. The data represent the mean value and SD. The difference was considered statistically significant at p < 0.05.Unless specified otherwise, statistical analysis of experimental data present in this paper were performed with Student's t test and ANOVA, with Tukey's multiple comparison test as To study whether sulfatide treatment shows pro-inflammatory or anti-inflammatory characteristics, we analyzed the secretion level of a well-known DAMP molecule, HMGB1. When treated simultaneously, sulfatide exhibited an inhibitory effect in HMGB1 secretion without toxicity in a dose-dependent manner, as shown . This phAlthough multiple points of inhibition are potentially available throughout the HMGB1 secretion pathway, they can be categorized into two large categories: initial signal transduction, and the release step. In order to clarify whether sulfatide affects the former or the latter, we treated Raw 264.7 cells with LPS or sulfatide and investigated HMGB1 localization via immunofluorescence microscopy . ConfocaPrevious reports state sulfatide to play a pro-inflammatory role in brain-resident immune cells . In ordeThe pathway most frequently associated with TLR signaling, NF-κB signaling pathway, is a cascade of signaling molecules that results in the degradation of NF-κB inhibitory molecules and the translocation of NF-κB to the nucleus, acting as a transcription factor. Concerning this pathway, we performed immunofluorescence microscopy, tracking the location of the p65 molecule, and immunoblotting of the IκBα molecule . Our immFurther analysis of the MAPKs within the TLR signaling pathway revealed specific kinases affected by sulfatide treatment. The phosphorylation levels of ERK, JNK, and p38 MAPK were analyzed via immunoblotting . ImmunobSince an alternate mechanism exists, where HMGB1 release can be triggered via LPS-TLR4 signaling through ROS production, we sought to measure the changes in the level of intracellular ROS in the presence/absence of sulfatide pre-treatment . Flow cyWe hypothesized that sulfatide, a well-known component of the cell membrane, may interfere with the lipid composition of the cell membrane, inhibiting its signaling pathways. Since TLR4 requires its monomers to be localized within the lipid raft microdomains to form dimers, we sought to assess  whether in vivo murine models were measured. BMDMs of 8 weeks old female C57BL/6 mice were harvested and were subjected to the same stimuli used above  mRNA  mRNA . Since TOS) mRNA , 47. FurOS) mRNA .1 receptor, are ascribed to multiple vascular diseases, such as hypertension and secondary cardiac hypertrophy . Lipid microarrays showed specific antibodies against various lipids in the cerebrospinal fluid, including ones against sulfatide in the murine EAE model and in multiple sclerosis , 58. KanOur research was able to report the anti-inflammatory effect of sulfatide in the periphery, specify the kinases within the NF-κB and MAPK pathway affected by sulfatide, and elucidate its mechanism of action. Sulfatide, nevertheless, is naturally a mixture of varying lengths of carbon chain backbone; therefore, the sulfatide used in this experiment is close to its natural form but far from being homogenous. Such properties could control the accessibility of sulfatide isoforms to various molecules via steric hindrance and variation in affinity. The composition of sulfatide isoforms has been connected to MS prognosis, enabling physicians to differentiate remitting MS from progressive MS by studying the composition of sulfatide isoforms . AlthougIn conclusion, our study showed the effect of sulfatide in suppressing the secretion of HMGB1 under LPS stimulation, and its potential as anti-sepsis treatment. We have also firstly described the mechanism of inhibition where sulfatide inhibits the localization of TLR4 within the lipid microdomains, nullifying LPS-TLR4 signaling cascade. Further investigations regarding the interaction of exogenous sulfatide with lipid microdomains, importance of sulfatide isoform composition in various inflammatory diseases, and in-depth studying of isoform lipid biology are necessary to pursue future therapeutic applications of sulfatide.All datasets generated for this study are included in the article/The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center.HK performed the immunoblotting and ELISA analysis with regard to HMGB1 secretion and its mechanisms. MH and IP helped in the planning and execution of the animal experiments. CP aided with the overall experiments using BMDMs. MK and IP contributed to the detailed experimental planning. J-SS gave the final approval of the manuscript version and has overseen the project and provided overall guidance for the experimental design. 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.


Astragali Radix (AR), the dried root of Astragali Radix membranaceus (Fisch.) Bge. or Astragali Radix membranaceus (Fisch.) Bge. var. mongholicus (Bge) Hsiao, is a commonly used traditional Chinese medicine for the treatment of liver diseases. This study aimed to comprehensively evaluate the pharmacological action and explore the potential mechanism of AR on liver fibrosis. Rats were administered with carbon tetrachloride for eight weeks, followed by oral treatment with AR for six weeks. The efficacy was confirmed by measuring liver function and liver fibrosis levels. The underlying mechanisms were explored by detecting the expression of related proteins. AR significantly decreased the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), collagen IV (COL-IV), hyaluronic acid (HA), laminin (LN), and precollagen type III (PCIII). In addition, AR inhibited the deposition of collagen and the activation of hepatic stellate cells. Those data strongly demonstrated that AR alleviated liver fibrosis by CCl4. In order to illustrate the potential inflammatory, the mRNA levels of IL-6, TNF-α, and IL-1β were detected. Subsequently, immunohistochemistry analysis was performed to further verify the expression of type I collagen. Finally, the expression of key proteins in the inflammatory signaling pathway was detected. AR significantly reduced the expression of high-mobility group box 1 (HMGB1), TLR4, Myd88, RAGE, and NF-κ B p65 genes and proteins. In addition, western blotting showed AR decreased the protein expression of RAGE, p-MEK1/2, p-ERK1/2, and p-c-Jun. Taken together, our data reveal that AR significantly inhibits liver fibrosis by intervening in the HMGB1-mediated inflammatory signaling pathway and secretion signaling pathway. This study will provide valuable references for the in-depth research and development of Astragali Radix against liver fibrosis. Liver fibrosis is a complex pathological process and an intermediate link between various liver diseases including cirrhosis and liver cancer , 2. And Astragali Radix (AR), the dried root of Astragali Radix membranaceus (Fisch.) Bge. or Astragali Radix membranaceus (Fisch.) Bge. var. mongholicus (Bge) Hsiao, is a commonly used traditional Chinese medicine for the treatment of liver diseases [It is necessary to develop new drugs for treating liver fibrosis . Traditidiseases . And it diseases . In traddiseases . Althougdiseases , 11, thediseases .κB signaling to induce inflammation and aggravate liver fibrosis. Studies have shown that extracellular HMGB1 binding to RAGE could phosphorylate c-Jun to induce a significant increase in type I collagen secretion [collagen1a1 and collagen1a2 genes [Experimental and clinical studies have found that the high-mobility group box-1 (HMGB1), recently discovered damage-associated molecular patterns (DAMPs), is substantially enriched in patients with hepatic fibrosis , 14. HMGecretion , 17, whia2 genes ).4-) induced liver fibrosis in rats? What is the underlying mechanism? Is the mechanism related to downregulation of HMGB1?Based on previous studies, this study aims to provide answers to the following questions: Does AR have therapeutic effect on carbon tetrachloride- . Alanine amino transferase , aspartate amino transferase , collagen IV , hyaluronic acid , laminin , and precollagen type III  detection kits were provided by Nanjing Jiancheng Bioengineering Institute . The antibodies of phospho-c-Jun, phospho-MEK1/2, HMGB1, α-SMA, phospho-ERK1/2, and ERK1/2 were bought from Cell Signaling Technology (United States). The antibodies of MEK1/2 and COL-I were purchased from Abcam (United States) and the antibodies of RAGE and GAPDH were bought from ABclonal (China).In this study, colchicine was purchased from Xishuangbanna Banna Pharmaceutical Co. Ltd. . CClAR membranaceus (Fisch.) Bge.) was supplied by Beijing Lvye Pharmaceutical Co. Ltd. . Ten times of water was used for the first extraction of AR for 2 h. Then, eight times of water was used to extract for 1.5 hours. The extract of Astragalus membranaceus was dried into powder by freeze vacuum drying oven. The final yield was about 40.08%. The Q-TOF LC/MS analysis showed that the main components of AR extract included 3,4-dihydroxybenzyl aldehyde (6.17%), calycosin-7-glucoside (0.69%), armillaripin (10.31%), rhodomollein III (7.05%), phthalic anhydride (1.76%), clavatine (6.46%), pterosin B (7.46%), D-cathinone (2.65%), formononetin (15.4%), Andrographolide (2.57%), and anemarrhenasaponin-I (4.55%) [AR (the dried root of  (4.55%) .A total of 36 male Sprague Dawley rats weighting 180–200 g were obtained from the China Food and Drug Certification Research Institute (Permission No. SCXK (Jing)-2014-0013). Those rats were housed in the Central Animal Laboratory of the Fifth Medical Center of Chinese PLA General Hospital. The animals were maintained under controlled conditions of temperature 25°C ± 2°C, humidity 55% ± 5%, and with light and dark cycles.4 in a ratio of 1 : 1. The model group, AR  groups, and colchicine (as a positive control group) groups were given CCl4 diluent (2 mL/kg) twice a week for 8 weeks [All the animals were randomly divided into six groups with six rats in each group. Olive oil diluted CCl 8 weeks . At the  8 weeks . The conThe serum levels of ALT, AST, COL-IV, HA, LN, and PC III were measured. In brief, according to the manufacturer's protocols, the samples, 2,4-dinitrophenylhydrazine solution, and sodium hydroxide solution were added in turn. The final color was read at 510 nm, and the serum ALT and AST levels were determined according to the OD value. Similarly, according to the manufacturer's instructions, add the reagents in the kit in turn, read the final color at 450 nm, and obtain the serum levels of COL-IV, HA, LN, and PC III according to the OD value.μm in thickness were cut and stained with hematoxylin eosin (H&E) and Masson. The pathological changes in the liver tissues were observed under a Nikon microscope .Liver tissue was fixed in 10% neutral buffered formalin for more than 48 h. All the livers were eluted with gradient ethanol solution and fixed and embedded in paraffin. Tissue sections about 4-5 α, IL-1β, HMGB1, TLR4, Myd88, and RAGE in liver tissues. Total RNA was extracted from 90 mg frozen liver tissue by Trizol reagent . The absorbance of the extracted mRNA was detected by spectrophotometer at 260 nm and 280 nm, respectively. The mRNA with purity (A260/A280) between 1.8 and 2.2 was selected as the qualified product for the follow-up study. RNA (2 μg) was transcribed into cDNA by PrimerScript RT regent kit . The cDNA was subsequently subjected to PCR amplification by ABI 7500 Real-Time PCR machine . Data analysis was performed by the 2−△△CT method. The primers are listed in Quantitative RT-PCR was used to detect the mRNA expression of IL-6, TNF-κB p65 , RAGE , phospho-MEK1/2 , MEK1/2 , phospho-ERK1/2 , ERK1/2 , phospho-c-Jun , and GAPDH , were used at this step. GAPDH was used as control for total protein extract. After incubation with appropriate secondary antibody for 2 hours at room temperature, the membrane was washed 3 times in TBS with 0.05% Tween 20 for 5 minutes each. Finally, the chemiluminescence system was used to measure immunoreactive proteins. All the western blotting was repeated 3 times.The mixture of ice-cold radio immunoprecipitation assay (RIPA) lysis buffer, phenylmethylsulfonyl fluoride (PMSF), and protein phosphatase inhibitor was used for protein extraction. Liver tissue (80 mg) was homogenized and lysed in the mixture, and then centrifuged at 12000 × g and 4°C for 10 min. The supernatant was collected and BCA Protein Assay kit  was used to detect the total protein concentration. All protein samples were levelled to the same concentration. Then, reducing Laemmli SDS sample buffer was added and mixed well, and boiled in boiling water for 5 minutes. The prepared samples were western blotted with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the blot was transferred to the polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was sealed in 5% skimmed milk at room temperature for 2 hours and then placed in a mixture of 5% skimmed milk, Tris-buffered saline (TBS), and 0.05% Tween 20 with the corresponding primary antibody, and incubated overnight at 4°C. Antibodies, including rabbit Anti-NF-α-SMA, HMGB1, and collagen I in liver tissue of fibrosis rats, immunohistochemical analysis was performed as described previously [α-SMA , Rabbit Anti-HMGB1 , and collagen type I  in a PBS solution at 4°C overnight. After incubating the sections with appropriate secondary antibodies for 1 hour at room temperature, they were stained with DAB staining solution and hematoxylin, and finally the slides were dehydrated and mounted. Nikon microscope  and NIS-Elements  software were used to observe the sections. Finally, Image-Pro Plus  software was used to calculate and analyze the average integrated optical density (IOD) of the positive area of the immunohistochemical reaction image.To evaluate the effects of AR on the expression of eviously . ParaffiP < 0.05 was considered statistically significant and P < 0.01 was considered highly significant.Results were expressed as mean ± standard deviation (SD). Data were evaluated by one-way ANOVA and Duncan's multirange test with the SPSS computer program . P < 0.01) compared with those in the control group. And serum ALT and AST concentrations were significantly reduced after oral administration of AR 5.4 g/kg and 10.8 g/kg (P < 0.01). Subsequently, the serum levels of COL-IV, HA, LN, and PC III which could reflect the degree of hepatic fibrosis were examined [P < 0.01). The inhibitory effect of AR on the levels of COL-IV, HA, LN, and PC III is dose-dependent. In particular, the serum levels of COL-IV, HA, LN, and PC III were significantly decreased in the 10.8 g/kg AR group (P < 0.01).As shown in Figures examined . As show4-treated rats showed partial necrotic in their liver accompanied by severe steatosis and inflammatory cell infiltration around the central vein of hepatic lobules. In contrast, the degree of liver damage, steatosis, and inflammatory infiltration in the AR treatment groups were reduced to varied degrees in a dose-dependent manner  staining and Masson staining were used to observe the pathological changes of liver tissue , 23. Thet manner . In the t manner . The aboα can promote the survival of activated HSCs and promote the development of liver fibrosis [β in patients with liver fibrosis caused by schistosomiasis is significantly increased [α, and IL-1β in rats by quantitative RT-PCR. The mRNA levels of IL-6, TNF-α, and IL-1β were increased remarkably in the CCl4-induced group compared with the control group . The results showed that AR had therapeutic effect on CCl4-induced liver inflammation in rats.Clinical data suggest that inflammation is a key factor in the progression of liver fibrosis . IL-6, afibrosis . It is rncreased . TherefoκB signaling pathway is one of the important pathways for liver aseptic inflammation. Therefore, we tested the expression levels of HMGB1, TLR4, Myd88, and NF-κB p65, which are the key targets of this signaling pathway in liver fibrosis rats. Quantitative RT-PCR and immunohistochemistry (IHC) methods were used to detect the expression and subcellular localization of HMGB1. As shown in Figures P < 0.05, P < 0.01).Literature research found that HMGB1, a cytokine abundantly enriched in patients with liver fibrosis, mediated TLR4//NF-P < 0.01). After treatment with AR, the expression of related genes decreased in a dose-dependent manner . Western blotting was used to detect the expression level of NF-κB p65 in the liver of rats with liver fibrosis. As shown in Figures κB p65 in the model group was significantly thicker than that of the control group, and the statistical results showed significant differences (P < 0.01). After AR treatment, the expression of NF-κB p65 decreased in a dose-dependent manner, especially in the 5.4 g/kg AR group and 10.8 g/kg AR group (P < 0.01). These results suggest that AR may improve liver inflammation by downregulating HMGB1/TLR4//NF-κB signaling pathway.Furthermore, the mRNA expression of HMGB1 was also quantified by using the RT-PCR method. The results showed that high dose of AR could significantly reduce the mRNA expression of HMGB1, which is consistent with the result of the IHC method. Those results proved that AR could reduce the expression of HMGB1 . Subsequα-smooth muscle actin (α-SMA) is a special marker of activated HSCs [α-SMA in the liver tissues  of the α-SMA-positive area of the AR treatment group was confirmed to be significantly reduced in a dose-dependent manner compared to model group (P < 0.01). Those results revealed that AR could reduce the activation of HSCs, a key cell of liver fibrosis.HSCs are the core cells of liver fibrosis. It is reported that HMGB1 can directly activate HSCs . Therefoted HSCs . Here, t tissues . In the 4 alone was significantly increased. After treatment with AR, the expression and deposition of type I collagen in the liver were significantly inhibited, especially in the group with the dose of 10.8 g/kg. The semiquantitative analysis of type I collagen immunopositive region further confirmed the above results (P < 0.01). It was previously reported that HMGB1 interacted with the receptor of RAGE to regulate the expression of type I collagen [4-induced group compared with the control group . Compared with the model group, the high-dose administration group of AR significantly reduced the expression of RAGE, p-MEK1/2, p-ERK1/2, and p-cJun .Next, IHC method was used to detect the expression of collagen type I. In Figures  results . The meacollagen . Therefo4-induced liver fibrosis model was performed in rats. Consistent with clinical observations, AR could improve liver function to some extent to alleviate liver injury [In this study, the aqueous extract of AR was used, which is consistent with the clinical use of traditional Chinese medicine. And the research results are more valuable. To determine whether AR has a protective effect on liver fibrosis, CClr injury , 29. In α, and IL-1β [β, TNF-α, and IL-6. It suggested that AR might improve liver fibrosis by inhibiting the inflammatory response in rats.Previous reports and the results of this study confirmed that AR has definite hepatoprotective effect and can effectively improve liver fibrosis , 30, 31.nd IL-1β , 33. Lonnd IL-1β . TherefoκB inflammatory pathway and promotes the transcription of inflammatory cytokines IL-1β, TNF-α, and IL-6 [κB p65. The results showed that the expression levels of TLR4, Myd88, and NF-κB p65 in AR treatment group were significantly lower than those in the model group. The above results suggest that AR might reduce CCl4-induced liver inflammatory by downregulating the expression of HMGB1/TLR4/NF-κB signaling pathway.Pathogen-free inflammatory liver disease is considered “sterile” . Studiesand IL-6 , 39. The4. The study also found that AR can not only downregulate the expression of HMGB1, but also reduce the transcription of inflammatory cytokine IL-1β, TNF-α, and IL-6 by regulating HMGB1/TLR4/NF-xB signaling pathway. Therefore, we speculate that AR can inhibit the activation of rat HSCs. The detection of α-SMA, a marker protein of HSCs activation, further confirmed our hypothesis: AR can significantly inhibit the activation of rat HSCs induced by CCl4  are recognized as the driving factors of liver fibrosis . It has  by CCl4 .Liver fibrosis or scar deposition caused by chronic injury is similar in all forms of liver disease . In the κB pathway, thus protecting rats against CCl4-induced liver fibrosis. Therefore, this study will provide scientific evidence and theoretical basis for the development of AR as a potential drug candidate for liver fibrosis.In conclusion, biochemical tests and histological assessments were used to reveal the function of AR. The study also demonstrated that AR was potent for downregulating the expression of HMGB1/RAGE/cJun pathway and HMGB1/TLR4/NF-


The addition of crumb rubber (CR) into base asphalt plays a critical role in the improvement of the performance of Asphalt-Rubber (AR) binders. However, due to the problems, like high constructing temperature and energy consumption brought by the additional rubber, the use of AR binders could be limited in some areas. During this study, CR is processed by microwave is adopted to reduce the viscosity of the AR binders system, while the CR processed by long screw extrusion also is studied. First, the swelling (the absorption of light component into the CR particle) and dissolution , both of which determine the improved performance of AR binders, are investigated by fluorescence microscopy and extraction tests. The size of the CR particle after swelling observed by fluorescence microscopy is used to evaluate the swelling rate of CR samples, and the ratio of the weight loss of CR samples after extraction to the original weight is employed to measure the dissolution rate. Then, Brookfield rotational viscometer and storage stability tests are conducted. Last, the rheologic performance, including high and low-temperature performances, is characterized by the dynamic shear rheometer (DSR) and bending beam rheometer (BBR), respectively. The fluorescence microscopy and extraction results show that microwave processing could effectively increase the swelling and dissolving rate, with the figures rising twofold and more than threefold, respectively. The results show that microwave processing could effectively reduce the viscosity of AR binders, with a viscosity decrease of 65% at 190 °C and, at the same time, the high temperature of Performance Grade (PG) decrease from 88 °C to 76 °C. The storage stability could be negatively impacted, but it is slight and the low-temperature performance is improved slightly. Asphalt Rubber (AR) generally refers to the mixture containing crumb rubber (CR), which is obtained by grinding the recycled crumbs of tires with base asphalt . CompareThe improvement of the performance of the AR binders is thought to be mainly coming from the adding of CR ,11. ActuThe swelling of CR could enhance both PE and IE, since it has the light absorbance component of asphalt, one of the main parts of forming IE, and increases the volume of CR particles, which has a positive impact on PE ,14. AddiThe preparation also could be an important process which decides whether the expected performance of AR binders can be obtained or not and, during this process, the main concerning factors include mixing types, time and temperature ,27,28,29(1)To measure the effects of microwave processing on CR swelling and dissolution;(2)To evaluate the viscosity and storage stability of AR binders after microwave processing;(3)To study the rheologic properties of AR binders after microwave processing.AR binders with great performance, however, are strictly used in some areas due to the extremely unpleasant odor and high constructing temperature caused by high viscosity. To reduce the viscosity of AR binders, the microwave is adopted in this study to process the CR without seriously influencing other properties. The main objectives of this paper are as follows:During this study, one neat asphalt binder (SK90) was used, sourced from Korea, and was labeled as 90# based on penetration grades. Three crumb rubbers were utilized: the first came from Hebei province, China, presented as CR-1; the second sourced from Shanghai city and was processed by long screw extrusion in factory for desulphurization, named as CR-2; the last was obtained by microwave-processing CR-1, reported as CR-3. Correspondingly, the AR binders composed by these CR were named as AR-1, AR-2, and AR-3. The size of all three types of CR was mesh #40. The basic information of the base asphalt is shown in The method that utilizes microwaves to process the CR was to desulphurize it so the viscosity of the AR binders could be reduced. The main procedure follows. Some CR was put into the oven at 60 °C for 60 min to dry the CR; about 60 grams of the CR was then sent to the microwave set at maximum power during desulphurization for 3 min. The Galanz G80F20CN2L-BB microwave was used in this study had a maximum power of 850 W and a frequency of 2450 MHZ, taking into consideration that the processing time should not be too long to prevent ageing and degradation of CR. During this study, all AR binders were prepared by the following procedures. First, about 500 grams of SK90# was sent into the steel container for oil bath heating to 170 °C; next, 20% by the weight of the base asphalt of CR was put into the base asphalt, slowly mixing at 100 r/min until all CR was added before the mixing speed was increased to 500 r/min; last, the mixtures were mixed for about 90 min at 180 °C and, after that, the end product AR was finally produced. During this study, the fluorescence microscope was used to measure the size of CR particles after swelling with the magnification of 200×, the size of which after swelling was regarded as the indicator to evaluate the swelling ability of the CR particles. The volume of CR particles was not measured due to the difficulty of finding the same CR particle before and after swelling. The typical scanning picture by fluorescence microscope is shown in 1 and m2 before the binders were tightly wrapped by the filter paper; next, about 200 mL toluene was poured into the extractor after which the extractor was assembled quickly in case of volatilizing of the toluene; then, the extractor was heated until the toluene in the extractor turned colorless and, subsequently, the AR binders wrapped by filter paper were removed and the AR binders after extraction were weighed and labelled as m3; last, the testing was repeated while the AR binders were replaced with CR and named as m4, which was to obtain the dissolving rate (the mass loss) of CR into toluene. The dissolving rates of CR into the base asphalt and into toluene were named as W and W0, respectively, both of which were calculated according to Equations (3) and (4).ε represents the crumb rubber dosage in the Asphalt-Rubber (AR).To measure the mass loss of CR, which dissolve into the base asphalt, the Soxhlet extraction method with toluene as solvent was utilized. The main testing procedures were as follows. First, about 2 grams of AR binders and a filter paper were weighed and named as mAn NDJ-1C Brookfield rotational viscometer was employed to obtain the rotational viscosity of binders at 130 °C, 150 °C, 170 °C and 190 °C, respectively, following the JTG E20-2011 T0625-2011, Standard Test Method of Bitumen and Bituminous Mixtures for Highway Engineering (China).Practically, storage stability is an important property which determines whether the great performance of binders can be obtained. The separation test was used to evaluate the storage stability of AR binders based on ASTM D7173. During the test, about 50 ± 0.5 grams of AR binders were put in an aluminum tube with the tube kept at 163 °C for 48 h, after which the tube was moved out and the softening point difference between the AR binders at the top and bottom of the tube was used to indicate the storage stability. A Dynamic Shear Rheometer  was employed to measure rheologic properties. The temperature sweep tests were conducted at the temperature range of 30–80 °C and data including the complex modulus, phase angle and rutting factor were obtained following the AASHTO T315-06. The applied strain was 10% with the frequency of 10 rad/s, and 25 mm parallel plate geometry was used during the testing. Also, the high-temperature grading could be determined based on results of rutting factors.τ is the applied creep load (0.1 and 3.2 kPa). Multiple stress creep and recovery (MSCR) tests are utilized to evaluate the high-temperature resistance to rutting of asphalt binders, following AASTO T350. During a standard MSCR test, it includes two parts with the applied force of 0.1 kPa and 3.2 kPa, respectively. The first part contained 20 load cycles and the second part had 10 load cycles. During every load cycle there was 1 s for applying force and 9 s for resting, which simulated the vehicle load being applied on the pavement. After testing, parameters of the recoverable rate (R) and non-recoverable compliance (Jnr) were calculated according to Equations (5) and (6). Also, research showed that Jnr was well related to the performance of anti-rutting at high temperature.Bending beam rheometer (BBR) was applied to characterize the low-temperature properties of asphalt binders, and the results of stiffness and m-value were calculated following ASTM standard D6648-08. To prepare the samples for BBR testing, the asphalt binders were heated to 160–180 °C to obtain enough mobility of the binders, then the hot asphalt binders were poured into the mold. Subsequently, the mold was put into cold water for 10 min before being demolded; last, the sample for testing was kept in a methanol bath at testing temperature for about 60 min. All samples were tested under −12 °C, −18 °C and −24 °C temperatures and the stiffness and m-value were calculated on a load time of 60 s.The volume of CR particles after the same swelling time and under the same experimental process was adopted during this study to evaluate the swelling rate of different types of CR. The reason for adopting the volume of CR particles after swelling is the difficultly in finding and observing the same CR particle before and after swelling. Meanwhile, the initial size of CR-1, CR-2 and CR-3 were in same level (mesh #40). During the test, a fluorescence microscope at the mode of magnification of 200× was used to observe the samples, and the single swelling particle in the observation area was chosen to calculate the volume of particles. The swelling rate of different CR were evaluated by the average of ten observation values. The results of the swelling rate are shown in It is clear from Based on the extraction experiments, both the CR dissolution rates in the base asphalt and in toluene could be obtained. It should be noted that the mass loss of AR after extracting includes molecules dissolving in asphalt and toluene, so the CR dissolution rate in the base asphalt is equal to the difference value of the mass loss rate and CR dissolution rate in toluene. Both CR dissolution rates in toluene and in the base asphalt results are shown in The CR dissolution rate in toluene was about the same level no matter whether the CR was desulphurized or not. This is mainly because the solvent extractive and natural rubber of CR are the two main substances that dissolve in toluene so the desulphurization process would not have an obvious change on the content of these two substances in CR. However, for dissolution rates in the base asphalt, the desulphurization would have a significant effect on this figure, with it rising more than threefold and twofold after microwave and long screw extrusion processing, respectively, compared with CR-1. Thus, it can be concluded that the desulphurization could increase dissolution rates in the base asphalt, which is great for IE. To conclude, the desulphurization could enhance both the absorption of the oily substance of the base asphalt and the molecules of CR dissolving into the base asphalt. The substance exchange between CR and the base asphalt can be accelerated by desulphurization and, as a result, the PE and IE can be established more quickly.The fundamental properties, including softening point, ductility, penetration, penetration index and elastic recovery, can be used to characterize AR binders preliminarily. The results are presented in It is clear from Storage stability of modified asphalt is an issue that always concerns both the researchers and constructors. The modified asphalt with poor storage stability cannot reach the expected performance when kept in high-temperatures for a relatively long time. Concerning polymer and the organically modified asphalt, poor storage stability can lead to modifier segregation, which could cause degradation of modifiers in an accelerated way. Many measures, like heating, high-speed shearing, adding compatibilizer and so on, are adopted to improve the compatibility between modifiers and the base asphalt . During The difference value of SBSMA, as a control group, was much higher than the others’, reaching more than 10 °C. During the heating process, the skin-like structures may be found in the surface, indicating the SBS modifiers segregate and float upward to the surface. All the AR samples have comparatively better storage stability. The figure for CR-2 was highest (5.9 °C), while the figures for CR-1 and CR-3 were only 3.1°C and 4.5 °C, respectively. It can be concluded that the storage stability of AR binders was not greatly influenced by microwave processing. It also should be noted that the softening points of AR in the bottom are bigger than those in the top, showing CR has a trend to sink downward.The viscosity of binders could not be too high during mixing and compacting processes, because higher viscosity means more energy needed to heat and obtain enough mobility and workability of the binder. So, to reduce the viscosity of AR, the microwave desulphurization process was adopted. The results of viscosity changing with temperature are presented in It is clear that the viscosity of all samples decreased with the temperature, and the viscosity of CR-3 was significantly lower than CR-1 and CR-2 at all temperatures. Meanwhile CR-2 also was consistently lower than CR-1. The figures for all AR binders, however, were still higher than SBSMA. Concerning how many mixing and compacting temperatures could be reduced after microwave processing, combination of the reduced viscosity and practical use in constructing still is required. It can be concluded that the desulphurization on CR could effectively reduce the viscosity of AR binders, especially using microwave processing. This conclusion also can be supported by other research that indicates sufficient depolymerization and devulcanization can result in the reduction of viscosity . */sinδ) combines the complex modulus and phase angle to evaluate the property of anti-rutting versus temperature. The data, including complex modulus, phase angle and rutting factor, are shown in The viscoelastic property of asphalt binders, including AR, vary greatly with temperature and is the main characteristic of rheology. The temperature sweep test was employed to measure the viscoelastic property from 30–80 °C. Complex modulus is an indicator of the stiffness and elastic component of materials. Occurring at high temperatures, the main challenge facing asphalt pavement is rutting, so a higher complex modulus is preferred due to its better anti-deformation performance. Conversely, the phase angle could indicate the viscous component of the materials, and a high phase angle (high viscous component) may indicate a poor anti-deformation. The rutting factor The swelling results showed that the swelling rate of CR-3, processed by microwave, was two times as high as CR-1’s (without processing). Meanwhile, the sizes of CR-1 and CR-2 particles were close. Considering the extraction test results, the dissolving rate in the base binder of CR-3 increased more than two times as compared with CR-1, and the dissolution rate of CR-2 was also one time bigger than CR-1’s. So, the substance exchange between CR and the base asphalt could be accelerated by desulphurization, especially by microwave processing.(2)The viscosity results showed that the viscosity of CR samples after desulphurization was consistently lower than CR-1’s at all temperatures and, especially, microwave processing had a noticeable effect of reducing viscosity (a viscosity decrease of 65% occurred at 190 °C). The storage stability result showed that the storage stability would be negatively affected by desulphurization, but it was still better than that of SBS-modified asphalt.(3)The high-temperature performance results showed that the high temperature PG grade decreased from 88 °C to 76 °C and the Jnr parameters of 0.1 and 3.2 kPa increased more than fivefold and threefold, respectively, after microwave processing, indicating that the microwave processing CR would exert a negative impact on the high-temperature anti-rutting performance of AR binders. Additionally, BBR results showed that the stiffness and m-value decreased and increased slightly at all temperatures, respectively, after microwave processing, indicating the low-temperature anti-cracking performance is improved. Also, low-temperature results of all AR binder samples performed better than SBS modified asphalt.During this study, the microwave processing of CR was utilized to reduce the viscosity of AR binder systems. Desulphurization of CR was achieved by breaking S–C and S–S bonds without the C–C bonds by controlling the maximum power of the microwaves and the working time. Then, the binding performance of AR binders was evaluated regarding its storage stability, viscosity, high-temperature anti-rutting and low-temperature resistance to cracking. The main conclusions of this research are summarized below:


Allergic rhinitis (AR) is a global disease that affects a huge proportion of people around the world especially in the Middle East, where multiple allergy-promoting factors can be found. Although AR is not fatal, it severely affects the quality of life. However, it is usually overlooked in developing countries due to resource scarcity. Findings. This study included 968 subjects with 721 (74.5%) females. The mean age was 24.69 years with AR prevalence at 47.9%. AR was associated with male gender [P = 0.001 ], having a job [P = 0.049 ], the having a chronic medical condition (P < 0.0001) mainly other allergies [P < 0.0001 ] and asthma [P = 0.006 ], using medications (P < 0.0001) and living in particular provinces (P = 0.010). However, no significant correlation was found with type of work and war factors except being distressed by war sounds [P = 0.027 ]. Finally, no associations were found with age, consanguinity, SES, educational level, and cigarette or/and shisha smoking (P > 0.05). Interpretation. Approximately half of the sample displayed AR symptoms, indicating a potentially high burden of AR in the community. A correlation to being distressed from war noises was found with AR which could reflect a psychological aspect. In addition, in war harmful allergens are released which can be an additional AR risk factor which adds to the environment in the Middle East that is associated with AR. However, we need further studies to discover and minimize this huge prevalence of AR. An online questionnaire on social media was used which included demographics, smoking, socioeconomic-status (SES), war-related questions, and the score for allergic rhinitis (SFAR), a simple self-reporting tool with the cut-off point at 7.  Rhinitis is a respiratory disorder in the upper respiratory airway, characterized by rhinorrhoea, itching, sneezing, and nasal obstruction. Allergic rhinitis (AR) and nonallergic rhinitis (NAR) are the classifications that were previously used according to the clinical manifestations and allergic sensitization to common allergens . AR occuWe conducted an online cross-sectional study in Syria from 26/03/2019 to 22/04/2019. Online Arabic surveys were used that included subjects who lived in Syrian provinces. We posted the questionnaires online twice each day at 10 AM and 10 PM in several groups that covered different topics such as educational, cuisine, merchandise, entertainment, cultural, and musical. Any participant who lived in Syria, agreed on participating in the study, answered the basic demographic questions, and AR questionnaire was enrolled in this study. No further criteria was applied as it is an epidemiological study.Socioeconomic status (SES): SES was assessed through three questions: the education of the person or the working family member, monthly family income, and the profession. As a result, SES was divided into 5 different categories: lower, upper-lower, lower-middle, upper-middle, and upper.Allergic rhinitis: We used an Arabic version of the score for allergic rhinitis (SFAR), a simple self-reporting tool with a cut-off point of 7 , 15. SFAWar-related questions: We asked several questions, both directly and indirectly, about the war including changing place of residence due to war, losing someone close, and being distressed by war noises.Smoking: We only assessed the current smoking status for cigarettes and shisha as getting more details would be beyond this study goal. We only asked two questions in this regards, “do you regularly smoke cigarettes” and “do you regularly smoke shisha”. We did not assess for individuals who quitted smoking and the amount and time of smoking.Other questions: We asked basic demographic questions including gender, age, educational level, province of residency, and having consanguineous parents. We asked the participants to declare having any medical condition and whether they used to take any medication. Age was divided into groups according to the national classification that was approved by Damascus UniversityOther definitions: Respiratory disease in this study was indicate to chronic bronchitis and chronic obstructive pulmonary disease (COPD). We defined low educational level as having a high school degree or less.Informed consent was taken from the participant before proceeding with the survey for participating in the research and for using and publishing the data. Confidentiality was assured and no questions indicative for the person were asked.Our study protocol and ethical aspect were reviewed and approved by Damascus University deanship, Damascus, Syria.P values were considered statistically significant.Data was processed using IBM SPSS software version 26 for Windows . Chi-square and one-way ANOVA tests were performed to determine statistical significance between the groups. Pearson's correlation was also calculated. We calculated odds ratios (ORs) and the 95% confidence intervals for the groups using the Mantel–Haenszel test by using the same software. Values of less than 0.05 for the two-tailed P > 0.05).Our study included 968 subjects with 247 (25.5%) being male and 721 (74.5%) being female. The characteristics of the subjects are demonstrated in P = 0.001 ]. Having a job was also correlated with having AR more frequently [P = 0.049 ]. However, it was not correlated with any type of work (P > 0.05). Having other medical conditions was also correlated with having AR (P < 0.0001), especially with having other allergic reactions [P < 0.0001 ] and having asthma [P = 0.006 ]. This was also the case with being distressed from war noises as it was correlated with having AR more frequently [P = 0.027 ].Comparing subjects with positive or negative AR is demonstrated in P < 0.0001). However, we did not find a statistically significant difference when comparing having AR with age, consanguinity, SES, educational level, cigarette and shisha smoking, and losing someone or changing place of living due to war (P > 0.05). When Pearson's correlation was calculated, no correlation was found between SFAR scores and age, SES scores, and the number of times of changing place of living due to war. No correlation was found with SES classification and SFAR scores when one-way ANOVA was used (P > 0.05).Furthermore, subjects who had AR took more medications, either prescribed or OTC  and the province of currently living (P = 0.010) with Idlib, Daraa, and As-Suwayda having less AR but Tartus and Deir ez-Zur as provinces of origin having more AR. Furthermore, Hama, Idlib, Homs, Daraa, and As-Suwayda as provinces of current living had less frequency of AR . The high prevalence of AR in Syria could be due to the war as it exposed the population to various substances. We found a statistically significant correlation between AR and distress from war noises. However, this estimation was based on a screening tool rather than a medical diagnosis due to lack of resources, especially at this time of conflict.AR has been previously categorised as perennial, intermittent, and occupational. However, the new classification is having symptoms either intermittently or persistently –18. We fIn a regional country close to Syria, there was no statistical difference between having AR and gender, smoking, place of living, and other housing and economic conditions . This waAnother study found that the coexistence of asthma and AR was more common in female adolescents . AnotherIn contrast, being employed increased the prevalence of AR in our subjects. However, no significant association was found with any particular type of employment. We also found a significant difference in AR prevalence depending on the province of residence. AR was found to increase the risk of asthma and atopic diseases.When AR is not aggressively and early treated, it can lead to an increase in the incidence of asthma . This waWhile rates of diagnosis and treatment appear to be high worldwide, there are few reports on patient satisfaction and potential unmet needs in AR sufferers. There is also a need for more data on the diagnosis of respiratory allergies, the use of symptomatic medications, and the potential role of a guideline-recommended treatment option, for example, allergen immunotherapy (AIT) , 45, 46.AR has complicated interactions with different risk factors and increases the risk of asthma if left untreated. It can also severely affect the quality of life. AR has a particularly high prevalence in the Syrian population which suggests underlying factors leading to this incline. This evidence reinforces the importance of studying AR and its risk factors, particularly in Syria, in order to have a comprehensive approach to treatment for such a prevalent health issue which contributes a significant burden on the health sector.This study was online based and, therefore, could not exactly determine the studied population. AR diagnosis was based on a screening tool, not a medical diagnosis. However, the effect of these two issues was minimized by the sample size and the high prevalence of AR found as it is a reflective result of the problemSmoking could only be simply addressed and correlated with ARNo further details could be collected on SES and war-related events as this may raise some concerns and it would not be accepted by Damascus University ethical commutee as it will not be nationally acceptable questions. SES measurement is also different from other countries as the average income and standards are different,