Stanford type A aortic dissection (TAAD) is one of the most life-threatening cardiovascular emergencies with high mortality and morbidity, and necroptosis is a newly identified type of programmed cell death and contributes to the pathogenesis of various cardiovascular diseases. However, the role of necroptosis in TAAD has not been elucidated. This study was aimed at determining the role of necroptosis in TAAD using bioinformatics analyses.  The RNA sequencing dataset GSE153434 and the microarray dataset GSE52093 were obtained from Gene Expression Omnibus (GEO) database. Differentially expressed genes of necroptosis (NRDEGs) were identified based on differentially expressed genes (DEGs) and necroptosis gene set. Gene set enrichment analysis (GSEA) was applied to evaluate the gene enrichment signaling pathway in TAAD. The STRING database and Cytoscape software were used to establish and visualize protein-protein interaction (PPI) networks and identify the key functional modules of NRDEGs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of NRDEGs were also performed. Additionally, Spearman correlations were used to construct the necroptosis-related transcription factor-target genes regulatory network, immune infiltration patterns were analyzed using the ImmuCellAI algorithm, and the correlation between immune cell-type abundance and NRDEGs expression was investigated. The expression levels of NRDEGs and immune infiltration were additionally verified in the GSE52093 dataset.  We found that the necroptosis pathway was considerably enriched and activated in TAAD samples. Overall, 25 NRDEGs were identified including MLKL, RIPK1, and FADD, and among them, 18 were verified in the validation set. Moreover, GO and KEGG enrichment analyses found that NRDEGs were primarily involved in the tumor necrosis factor signaling pathway, nucleotide-binding oligomerization domain-like receptor signaling pathway, and interleukin-17 signaling pathway. The imbalance of Th17/Treg cells was identified in the TAAD samples. Furthermore, correlation analysis indicated that expression of NRDEGs was positively associated with proinflammatory immune-cell infiltrations and negatively associated with anti-inflammatory or regulatory immune-cell infiltrations.  The present findings suggest that necroptosis phenomenon exists in TAAD and correlates with immune cell infiltration, which indicate necroptosis may promote the development of TAAD through activating immune infiltration and immune response. This study paves a new road to future investigation of the pathogenic mechanisms and therapeutic strategies for TAAD. Aortic dissection (AD) is one of the most common catastrophic cardiovascular diseases caused by tearing of the intima with consecutive bleeding into the media of the aortic wall , 2. AccoImmune infiltration has been the hot spot in the field of cancer in the recent decade, and a small but growing number of studies have shown that the immune-inflammatory pathway is involved in pathophysiological and metabolic alterations of AD , 6. Notaα and interferon-γ, trigger death receptors and recruit receptor-interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to compose a regulatory necrosome complex. Activated RIPK3 binds and activates the downstream executor mixed lineage kinase domain-like protein (MLKL) by phosphorylation at the serine 232 site. Phosphorylated MLKL shifts into an oligomerized state that facilitates the formation of membrane-disrupting pores, finally leading to necrotic death. Due to the rapid plasma membrane rupture, proinflammatory intracellular contents are released and subsequently induce an inflammatory response. Recently, accumulating studies have investigated necroptosis mechanisms involved in the pathogenesis of cardiovascular diseases, including aortic aneurysm and dissection [−/− mouse models of acute aortic aneurysm or dissection, and inhibition of RIPK3-mediated necroptosis could prevent aortic enlargement and acute aortic aneurysm or dissection formation. The findings indicate a vital role of necroptosis for necroptosis in the formation of aneurysms and dissection [Necroptosis is a newly identified form of programmed cell death with hallmark features of necrosis . Necroptssection –17. For ssection –20. HoweIn this study, we conducted a systematic bioinformatics analysis using the Gene Expression Omnibus (GEO) database to outline the immune infiltration landscape and determine whether and how necroptosis contributes to the development of TAAD. Moreover, we explored the potential functional mechanism and key genes of necroptosis in TAAD. Additionally, we deliberated the relationship between necroptosis and infiltrating immune cells to gain a better understanding of the potential molecular immunity process during the development of TAAD.The RNA-seq transcriptome data of patients with TAAD and the corresponding clinical data were obtained from GEO by R package “GEO query.” GSE153434 dataset  contains 10 AD ascending aorta tissues and 10 normal aortic tissues, which was performed on the GPL20795 (Illumina HiSeq X Ten Homo sapiens) platform . The datp < 0.05 [https://ggplot2.tidyverse.org) and the heatmap R package (https://CRAN.R-project.org/package=pheatmap). Eight necroptosis genes in the gene set M24779.gmt were retrieved from the Gene Set Enrichment Analysis (GSEA) (http://www.gsea-msigdb.org/gsea/index.jsp). Additionally, we collected the profiles of 159 genes related to necroptosis from the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway databases (https://www.genome.jp/dbget-bin/www_bget?pathway+hsa04217). Supplemental material SWe screened differentially expressed genes (DEGs) using the R language package “limma (v 3.48.3),” and the cutoff was adjusted p < 0.05 . Volcanohttps://string-db.org/) and Cytoscape software (version 3.8.2) [The PPI network of NRDEGs was visualized by the STRING database (n 3.8.2) , 24. Cytn 3.8.2) , 26.p < 0.05. Gene set variation analysis (GSVA) was used to evaluate the association between biological pathways and gene signatures based on expression profile data. The gene set “c2.cp.kegg.v7.5.1” for GSVA analysis was downloaded from the MSigDB database.To investigate potential biological functions between AD and normal control, Gene Ontology (GO) enrichment analysis and KEGG pathway analysis were conducted by R package clusterProfiler. Only terms with false discovery rate <0.05 were considered statistically significant enriched pathways. Moreover, the “clusterProfiler” package was used to perform GSEA for the potential mechanism of c2 (c2.cp.kegg.v7.5.1.entrez.gmt) and c5 (c5.bp.v7.5.1.entrez.gmt) in the molecular signature database (MSigDB) . The numTo investigate the molecular mechanism of necroptosis, the TRRUST, MSigDB, RegNetwork, and hTFtarget databases were utilized to identify TF-target gene interaction pairs and retrieve those involved in necroptosis regulation. The necroptosis-related transcription factor–target gene regulatory interaction network was generated, and the NRDEGs obtained from the GSE153434 dataset were used to construct the necroptosis-related TF-target regulatory network, which was calculated using Spearman correlations and visualized using Cytoscape (V 3.8.0). The lowest Spearman correlation coefficient of |0.6| demonstrates unequivocally that biologically significant gene correlations occur.http://bioinfo.life.hust.edu.cn/web/ImmuCellAI) [A comprehensive prediction of immune cell abundance was provided by the Immune Cell Abundance Identifier (ImmuCellAI) (uCellAI) . ImmuCelp value of < 0.05 was considered significant. Immune cell abundance in GSE52093 was also estimated using ImmuCellAI.The expressions of the NRDEGs were extracted from GSE52093, and the difference between TAAD and normal samples was calculated and visualized by package ggpurb and ggplot2 respectively. A We revealed a total of 2739 DEGs between TAAD and normal samples in GSE153434, with 1253 genes upregulated and 1486 genes downregulated. p value of 0.005, indicating an overall strong link between necroptosis and TAAD. GSEA also revealed that gene expression relevant to apoptosis, nucleotide-binding and oligomerization domain- (NOD-) like receptor signaling pathway, TNF signaling pathway, Th17 cell differentiation, and Toll-like receptor signaling pathway was significantly enriched in AD  alterations in the expression of RIPK1, MLKL, and other 23 genes; (2) activation of necroptosis in GSEA; (3) effectual construction of the TF-NRDEGs regulatory network. Furthermore, the enrichment analysis revealed that NRDEGs were primarily engaged in immune-inflammatory response pathways, including TNF signaling pathway, NOD-like receptor signaling pathway, and IL-17 signaling pathway. Additionally, we observed a dramatic difference in immune cell content between TAAD and normal samples. Intriguingly, both RIPK1 and MLKL were positively associated with macrophages and Th17 infiltrations and negatively associated with Treg cells. To the best of our knowledge, this is the first bioinformatics report describing the coexistence of necroptosis in the pathogenesis of human TAAD.The vascular smooth muscle cell (VSMC) loss is a prominent pathological characteristic of TAAD , 15, 29.α-mediated and Toll-like receptor- (TLR-) induced necroptosis have been widely investigated [α might be the primary factor regulating necroptosis. We also found that the vascular smooth muscle contraction pathway and muscle cell development pathway were inhibited, which partially explains the VSMC loss in TAAD. Intriguingly, multiple immune-related pathways were also enriched in AD samples, including B cell-mediated immunity, NOD-like receptor signaling pathway, Th17 cell differentiation, T-cell differentiation involved in immune response, and Th2 cell differentiation. These findings were further confirmed in subsequent immune infiltration analyses.In addition to necroptosis and apoptosis, GSEA also identified several other biologic pathways that were significantly and potentially relevant to TAAD. Notably, the TNF signaling pathway, which may promote extrinsic apoptosis and necroptosis, was enriched and activated in TAAD. TNFstigated . TLR sigIn the present study, we applied ImmuCellAI to estimate the abundance of 24 immune cells based on the microarray profiles of aortic tissues samples and obtained a comprehensive view of the immune infiltration landscape. The macrophage score was higher in TAAD; the finding is consistent with that in previous studies , 37. MacThe present study has several limitations. Firstly, although a rigorous bioinformatics analysis was conducted in the present study, our results need to be carefully assessed due to the lack of confirmation from our own experiments and clinical trials. Secondly, the sample size was relatively small, and the results of this study should be confirmed in larger prospective studies. Thirdly, immune infiltration analysis was performed based on transcriptomic data. Hence, we could not determine if necroptosis caused immune cell infiltration, or whether immune cells are also involved in the process of necroptosis. Further studies are warranted to clarify the underlying mechanisms.Bioinformatics analyses revealed that the expression levels of necroptosis-related genes significantly differed between TAAD and normal aortic tissue samples. Moreover, we found the imbalance of Th17/Treg cells and the correlation between the expression of ferroptosis-related genes and the infiltration of multiple immune cell types in TAAD. The necroptosis- and immune-related factors identified in our analyses may help determine the underlying mechanisms of AD pathophysiology and identify novel targets for medical interventions.


The purpose of this study was to clarify the vitamin D (VD) effect on male infertility. Our research was conducted using the following electronic databases: MEDLINE, Embase, Web of Science, Scopus, ClinicalTrials.gov, and Cochrane Library. Selection criteria included all published randomized controlled trials and non-randomized studies, focusing on vitamin D and male reproductive function. The results showed that the effects of VD on male reproduction has been investigated in three different topics: the molecular mechanism underlying VD effects on semen quality (SQ), the relationship between VD levels and SQ, and the effect of VD supplementation on SQ. Results supported the hypothesis of a relevant interrelation between concentrations of male VD and semen parameters, with particular reference to sperm motility; on the contrary, evidence on the effect of VD on male sex steroid hormone levels was inconclusive. The results of this review hold up the thesis that VD plays a role in male reproduction. Most of the data highlighted a positive effect on semen quality, particularly in sperm motility, both in fertile and infertile men. Additional dedicated studies are required to elucidate the still controversial aspects of this topic. Vitamin D (VD) is a group of liposoluble vitamins, synthesized in the skin due to Ultraviolet B (UVB) sun radiation; it has the characteristic to bind to a classical steroid hormone receptor, such as it was a “pro-hormone” ,2. VD plMore recently, VD has been defined as a “pleiotropic” molecule with several hypothesized actions and functions ,9. In thRecent in vivo data confirmed the association between VD, reproductive hormones and sperm parameters in infertile males, supporting the hypothesis that VD could have a positive effect on male reproductive capacity ,14. FurtTo better clarify the main aspects of the relationship between VD and male reproduction, we performed a systematic review of the inherent literature with particular attention to the effects of VD on male infertility.The research was conducted using the following electronic databases: MEDLINE, Embase, Web of Science, Scopus, ClinicalTrials.gov, and Cochrane Library. The studies were identified using combinations of the search terms “male reproduction”, “sperm”, “testis”, “gonad”, “fertility”, “epididymis”, “semen quality”, “sex hormones”, “testosterone”, in combination with “vitamin D”, “vitamin D levels”, “vitamin D receptor”, “vitamin D role”. We decided to limit the search to articles published from January 2010 to December 2020, in order to guarantee the relevance and up-to-dateness of the paper.Only scientific publications in English were included. Selection criteria included all published randomized controlled trials (RCTs) and non-randomized studies (NRSs) , focusing on vitamin D and male reproductive function. Two independent reviewers (G.C. and V.C.) conducted the review steps . Disagreements were resolved by consensus with a third reviewer (A.P.). All cross-references were also hand-searched. Proceedings of scientific meetings and abstracts were not considered. We excluded from the analysis the experimental in vitro studies or on animal models.All articles describing the effects of VD on SQ and/or male fertility were considered. Only original papers that reported specific experience data on the topic were included. All the relevant aspects of the included papers were recorded and commented. The review was reported following the Preferred Reporting Item for Systematic Reviews and Meta-Analyses (PRISMA) statement . Local Sn = 6) or animal studies (n = 4). Finally, 17 studies were included for the analysis [We found 1283 records. Twenty-seven articles were selected as possibly relevant. Ten studies were then excluded as they were review or comment articles not reporting original data ; b. the relationship between VD status and SQ; c. the effect of VD supplementation on SQ.VD molecular mechanism ,12,27;VD level and SQ ,29,30,31VD supplementation and SQ ,22,28.Based on these considerations, we divided the achieved included articles into sections, based on the specific topic:n = 13; epididymis n = 7), prostatectomy (prostate n = 5 and vesicles n = 3) and semen samples (n = 13), the authors found expression of VDR and VD metabolizing enzymes in germ cells during spermatogenesis, persisting in the ejaculatory duct and in ejaculated spermatozoa [Poor data exist on the VD metabolizing enzymes  expressed in human testis and the male reproductive tract. In 2010, Blomberg Jensen et al. demonstrated the expression of VDR and the VD metabolization in human testis and epithelia of the ejaculatory tract. Based on tissue samples obtained from orchiectomy  [Successively, in 2012, the same group of authors investigated the CYP24A1 enzyme expression as a possible marker of VD metabolism in spermatozoa and its association with SQ [Recently, Jueraitetibaike et al. explored sperm kinetic parameters in vitro after incubation for 30 min with 1,25 (OH)2 VD, verifying an increased upward migration of spermatozoa with growing adenosine triphosphate (ATP) levels . They noIn the last years, the reports of pandemic VD deficiency brought on the extension of the spectrum of extra-skeletal research on the topic and in particular more interest has been focused on the relationship between VD and SQ, with attention also on related sex hormonal values. All the main findings of this review on this topic are described in p < 0.05) was noted; in the case of VD deficiency (<25 nM), males had a lower proportion of motile (p = 0.027), progressive motile (p = 0.035) and morphologically normal spermatozoa (p = 0.044) in comparison with with high VD levels males (>75 nM). The authors reported an additional in vitro result: via VDR-mediated calcium release from an intracellular calcium storage, VD increased intracellular calcium levels in human spermatozoa rise the sperm motility and produce the acrosome reaction [n = 101) had approximately 31% lower total sperm count and 23% lower normal morphology percentage compared with low (n = 103) and medium (n = 103) VD levels [In a cross-sectional association study on 300 men from the general population, Blomberg Jensen et al. in 2011, aimed to show the role of activated VD in human spermatozoa and the hypothesis of association between VD serum levels and SQ . A positreaction . In the n = 19), VD < 20 ng/mL; group 2 (n = 108), VD > 20 and <50 ng/mL; group 3 (n = 20), VD > 50 ng/mL [p < 0.05). No statistical difference between the mean hormonal values in the different categories of VD was seen [In 2012, Hammoud et al. evaluated SQ parameters in 3 groups of healthy patients, with different serum VD concentrations: group 1 (50 ng/mL . The relwas seen .n = 195), infertile men (n = 9) with osteoporosis risk factors (WR) and infertile men (355) without osteoporosis risk factors (WOR) were enrolled; WOR men were divided into oligo-astheno-teratospermic  and non-obstructive azoospermic  groups. No correlations between T and VD were found in the examined groups. Focusing on the comparison between fertile and WOR-OATN men, a positive relationship between VD values and sperm motility as well as morphology in both study groups was found; in particular, OATN men with sufficient VD had a significantly higher percentage of morphologically normal spermatozoa than males with deficient (p = 0.005) and insufficient VD (p = 0.034) [In a cross-sectional study, Yang et al. aimed to investigate testosterone (T), VD, and SQ and their relationships in an infertile Chinese male population . Fertilep < 0.05) in couples with normal VD levels, as well as delivery rate  [The observational study of Tartagni et al. evaluated the negative influence of low male VD serum levels on the conception rate in couples attempting pregnancy . Ninety  < 0.01) .n = 186) and oligoasthenoteratozoospermic (OAT) (n = 92) men. VD levels revealed no association between sperm parameters and hormone balance in normospermic population; nevertheless, sperm motility was positive correlated with VD categorized in OAT men (p < 0.05) [In 2016, Abbasihormozi et al., compared VD and hormone levels in normospermic ( < 0.05) .n = 186) and fertile (n = 79) patients. However, VD levels had a positive statistically significant association with progressive motility and sperm count (p < 0.05) [In the case-control study of Zhu et al., analysis of serum inactive VD (VD-OH) value reported no significant difference between infertile ( < 0.05) .p =0.04) and 4 h (p < 0.0005) after ejaculation; on average, the numbers of motile or progressive motile sperm were inferior in VD deficient men (p < 0.05). No significant relationship between lower total testos and VD deficiency was found [A study comprising a baseline of 1189 infertile men, screened for the Copenhagen Bone-Gonadal (CBG) study, who underwent a physical examination, semen evaluation and blood analysis for serum VD and hormonal profiles . Positivas found .n = 23) had significantly higher VD levels (p < 0.001) as compared to impaired total sperm motility (n = 81) [p < 0.001) [A retrospective study by Tirabassi et al. showed the interconnection between serum VD and SQ parameters based on a case-series study of andrological patients. Normal total sperm motility males ((n = 81) . Authors(n = 81) . Akhaviz< 0.001) .p = 0.027), progressive motile (p = 0.035) and morphologically normal spermatozoa (p = 0.044) compared with men with high VD levels (186 controls). The median value of serum VD was significantly greater in the control group compared to the case group (p < 0.001); moreover, serum T level had a significant positive association with VD whereas luteinizing hormone (LH) had a significant negative association (p < 0.001). Limiting analysis to infertile subjects, high values of VD were observed in infertile samples with normal SQ (62.15 ± 13.86 nmol/L) in comparison to infertile men with altered SQ (51.87 ± 16.77 nmol/L) [In a cross-sectional study, Rehman et al. found that VD serum level seems to have a positive correlation progressive motility . The cohp < 0.03), but at day 150, there were not statistically significant difference about median sperm count and sperm concentration given the two study arms at day 150 (p = 0.07) [p < 0.07); interestingly, analysis of a sub-group of oligozoospermic men with VD implementation showed an increased possibility of a live birth compared with placebo [The Copenhagen Bone-Gonadal Study (CBG) is a randomized clinical trial carried out by Blomberg-Jensen et al., in which 330 infertile men with VD insufficiency (≤50 nmol/L) randomly received placebo or a high initial oral dose of VD dissolved in oil , followed by 1400 UI VD and 500 mg of calcium daily for 150 days, in order to compare the outcomes on SQ, reproductive hormones levels and live birth rate . Authors = 0.07) . However placebo .n = 52) [p = 0.047) and sperm motility increased from 22.2 to 27.8%. Moreover, oral VD supplementation improved sperm motility (5.6%), however, not in a statistically significant manner [p < 0.001). Finally, the overall study pregnancy rate was 8.33% after VD supplementation; however, this data was not statistically significant (p = 0.24) [In a pilot-study, Waud et al. evaluated if semen parameters could be improved with repletion of daily oral VD for 90 days in a male population with abnormal SQ (n = 52) . The meat manner . After a = 0.24) .In the last decade, interest in VD has grown, mainly after the identification of several “non-classical” targets of action, including the reproductive organs. The established expression of VDR and VD-metabolizing enzyme in the ovary, uterus, placenta and also in male reproductive tract and human spermatozoa, suggested an important role of VD for reproductive physiology regulation ,32,33,34Although it is a very interesting issue, our review of the international literature highlighted the presence of only a small number of articles on the role of VD in reproductive adulthood life and the fact that it has only been the subject of research in recent times ,37,38,39First of all, there are still few data on the mechanism of action of VD with reference to the male genital system and therefore several obscure points persist to this day. Starting from the evidence that the human testis and the ejaculatory tract are sites of metabolizing VD , the ideCertainly, the main object of our search was the correlation between male VD deficiency or insufficiency and characteristics of SQ. Although the correlation between VD, semen total count and morphology rate remain ambiguous, the results on association with sperm motility appear more significant. Most of the specifically related articles included in the review (9 out of 11 included articles) reported a positive association of VD serum level with semen total motility, both in fertile and infertile males; moreover, in many cases, this positive association of VD level and semen total motility had statistical significance ,26,29,31Another very interesting aspect of our research concerns the possible role of VD supplementation on SQ. Although overall results on the topic suggested that VD supplementation had no significative association with changes in semen parameters, data on the spontaneous pregnancy rate in couples with infertile men were surprising; in particular, the results of two interventional studies on infertile men with VD deficiency showed the increased chances of a spontaneous pregnancy in the population treated with VD and VD plus calcium supplementation ,28. ObviOverall, some limits should be taken into account in evaluating the results of our review. Literature on the topic is poor: in most studies, the sample size was small with a wide study design heterogeneity. Moreover, possible confounding factors  could make it difficult to correctly interpret the studies. For these reasons, the clinical importance of all our observations should be validated by further research.The results of this review support the hypothesis that VD plays a role in male reproduction. Most of the data highlighted a positive effect on semen quality, both in fertile and infertile male populations. Instead, evidence on any relationship between VD and sperm total count as well as morphology was inconsistent; moreover, research on the association between VD status and sex hormone production did not provide strong results. Experimental studies on the evaluation of VD levels and VD supplementation in men with VD deficiency or insufficiency are scarce to-date and it is not possible to draw conclusions; however, the preliminary data seem promising. This aspect might encourage a new point of view and a new perspective for infertile men and couples in their pregnancy search path. Further dedicated experimental studies are required to clarify the still controversial aspects of this topic.


Mytilus galloprovincialis and Argopecten irradians were used as experimental shellfish species for in situ monitoring. We compared the inflammatory-related gene responses of the two shellfish during PSTs exposure by using transcriptomes. The results showed that the accumulation and metabolism rate of PSTs in M. galloprovincialis was five-fold higher than that in A. irradians. The inflammatory balance mechanism of M. galloprovincialis involved the co-regulation of the MAPK-based and AMPK-based anti-inflammatory pathways. A. irradians bore a higher risk of death because it did not have the balance system, and the regulation of apoptosis-related pathways such as the PI3K-AKT signaling pathway were upregulated. Taken together, the regulation of the inflammatory balance coincides with the ability of bivalves to cope with PSTs. Inflammation is an important factor that affects the metabolic pattern of PSTs in bivalves. This study provides new evidence to support the studies on the resistance mechanism of bivalves to PSTs.Paralytic shellfish toxins (PSTs) are an increasingly important source of pollution. Bivalves, as the main transmission medium, accumulate and metabolize PSTs while protecting themselves from damage. At present, the resistance mechanism of bivalves to PSTs is unclear. In this study,  Alexandrium, Gymnodinium, and Pyrodinium in the marine environment. Among these, the Alexandrium species, which are known to produce paralytic shellfish toxins (PSTs), are the most widespread shellfish-contaminating biotoxins and have a worldwide distribution [A. catenella, A. tamarense and G. catenatum, etc., have been identified [Alexandrium [Alexandrium were established by cyst germination, and all three strains had the same toxin profile consisting of gonyautoxins 1, 3, 4  and neosaxitoxin (NEO) [Saxitoxin and its analogues  are a type of shellfish toxin produced by dinoflagellates such as ribution . In Chinentified ,3,4. An xandrium . During in (NEO) .Alexandrium exposure has a negative impact on survival and growth in some bivalve populations, by interfering with feeding activities and shell valve closure [Alexandrium exposure can also produce histopathological lesions, inflammatory responses and impair immune responses in bivalves exposed to them [Alexandrium minutum in M. galloprovincialis [ closure . Alexand to them ,9. Studi to them ,11. Gene to them . The use to them ,14. In c to them . Recent  to them . Transcr to them ,18, and  to them . The comincialis . Howeverincialis ,22,23. Iincialis ,25. HoweMytilus edulis. Moreover, a successful acute inflammatory response results in the elimination of the infectious agents followed by a resolution and repair phase, indicating that anti-inflammatory factors are crucial for the transition from inflammation to resolution [Perna viridis) have potential anti-inflammatory properties and have been marketed as nutraceuticals against arthritis [Bivalves as invertebrates lack adaptive immunity and rely primarily on the innate immune system for their defense ,27. Inflsolution . Mussel rthritis . As is wrthritis . Our undAlexandrium catenella bloom has occurred in Qinhuangdao [M. galloprovincialis was the cause of PST poisoning at a relatively high level of over 4.9 × 104 μg STX eq/kg in 2016. M. galloprovincialis and A. irradians are the main cultured species in this area. However, a previous study found that the exposure to PSTs caused large-scale mortality of bay scallops, while mussels were relatively unaffected (unpublished data). In order to examine the difference in resistance, we focused on inflammatory reactions. We exposed two shellfish species to PSTs during natural blooms from April to May and monitored the resulting content of PSTs. Samples from the elimination processes were collected for transcriptome analysis. The key pathways were revealed by comparing the stress differences between the two species from the same environment. The outcomes of this study will increase our understanding of bivalve–PSTs interactions and interspecific differences in the resistance to PSTs.Since 2016, a recurring Alexandrium catenatum red tide were examined by using the LC-MS/MS technique. During the experiment, the algal density of A. catenella was monitored as shown in M. galloprovincialis and A. irradians (p < 0.01) after toxigenic algae blooms  during the same period. Indeed, the accumulation of toxins in the scallop was low, and there was no rapid metabolism of the toxins. There were also significant differences in the accumulation ratio of each analogue  from the beginning of May. M. galloprovincialis displayed strong survivability and insensitivity to PSTs compared to A. irradians. According to the toxin elimination rate, we divided progress into fast (“Fast-stage”) and slow (“Slow-stage”) stages for transcriptome analysis.The shellfish collected from Qinhuangdao waters exposed to an e blooms a. PSTs aanalogue b. GTX1 wM. galloprovincialis and A. irradians, respectively. The Q30 values in the samples were greater than 95%  and down-regulation in the Slow-stage, which is consistent with the rule of toxin metabolism. The p38 pathway, in contrast, was complementary to JNK (MKK6). Genes related to the NF-κB pathway (NF-κB1 and IKBKB) were significantly upregulated in the Slow-stage of M. galloprovincialis; conversely, NF-κB1 was significantly down-regulated in the Fast-stage in A. irradians.Gene set enrichment analysis  can successfully compensate for the lack of effective information mining of micro-effective genes by traditional enrichment analysis, and it can explain the regulatory role of a functional unit (KEGG pathway or others) more comprehensively. In this study, GSEA analysis identified MAPK as the core conjunction module with interspecific and temporal differences. The RAS, TNF, and NF-κB signaling pathways also acted in coordination with MAPK, as shown in pathways . In M. gM. galloprovincialis, and not significantly down-regulated in A. irridians  [A. tamarense at a density of 1.26 × 104 cells/mL, the concentration in A. irradians was 49.4 MU/g (the regular standard is 4 MU/g) [A. irradians had stronger accumulation ability, but this could not be determined due to different environmental conditions. Therefore, M. galloprovincialis and A. irradians were placed in the same environment for experiments in this study. The opposite result appeared in our study, i.e., M. galloprovincialis exhibited higher PSTs tolerance as well as a stronger ability to accumulate and metabolize PSTs. In contrast, the slow metabolism and high mortality rate of A. irradians indicated weaker adaptability to the stress.A limited number of studies have shown that the ability to accumulate PSTs between 0 μg/kg) . After e 4 MU/g) . It seemM. galloprovincialis were five times higher than in A. irradians, reaching 5604.8 μg STX eq/kg. This may be caused by the individual specificity of the PSTs exposure of the two shellfish, and some studies support this conjecture. Studies have shown that the axons of M. galloprovincialis neurons were insensitive to STX [A. irradians is very sensitive to PSTs, leading to shell closure and reduced food intake [In this study, PSTs distributed in e to STX , so theyd intake .M. galloprovincialis was two times higher than in A. irradians, which may be related to their pan-genome [M. galloprovincialis, it is expected that some of the differential genes were not related to the response to PSTs accumulation, but constitute background noise. However, it has also been proposed that accessory functions provided by the large number of dispensable genes may underpin the improved ability to adapt to challenging and varying environmental conditions [M. galloprovincialis displays adaptive traits to maintain immune capacity upon prolonged PSTs exposure, including a selective evolutionary advantage [A. irradians displayed negative effects when faced with the same stress [M. galloprovincialis present stronger immune responses to PSTs exposure.PSTs induce oxidative stress, paralysis, and immune impairment of bivalves . In thisn-genome . This imnditions . The pubdvantage  and the dvantage . In conte stress , as immue stress , and unce stress . In summM. galloprovincialis but not in A. irradians. This is related to the stronger antioxidant capacity of mussels in the early stages [M. galloprovincialis. JNK activation is a key determinant in the cellular response. The activation of the JNK pathway was consistent with the metabolic rule of PSTs. Some researchers have suggested that JNK plays an active role in toxin stress [Available data on bivalve immunity through the omics approach have only been obtained in the last few years. The inflammatory response is likely to be involved in repairing damaged tissues  and in dy stages . In the n stress .A. irradians displayed immunosuppression after exposure to shellfish toxins such as okadaic acid [A. irradians showed non-significant and significant inhibition, which may indicate the immunosuppression of A. irradians against PSTs as well, but the immunosuppression indicators still need to be measured. Continuous upregulation of signal transduction activated the NF-κB1 in the Slow-stage of M. galloprovincialis, while this pathway was significantly down-regulated in the Fast-stage in A. irradians. Therefore, the two species are diametrically opposite in terms of cell survival and apoptosis regulation. Therefore, an unexpected finding was that the PI3K-AKT signaling pathway was significantly upregulated in the transcriptome of A. irradians at the Slow-stage. Additionally, the PI3K-AKT signaling pathway has previously been shown to have a synergistic effect with MAPK in promoting the inflammatory reaction [In contrast, aic acid , domoic aic acid , and micaic acid . In thisreaction . To summM. galloprovincialis, suggesting that AMPK could inhibit MAPK. The metabolic regulation process of mussels for toxins is more diversified. As a result of the self-limitation of the p38 MAPK signaling pathway and activation of the AMPK signaling pathway to inhibit the MAPK/NF-κB pathway, damage to the body of the mussel is significantly reduced through a dynamic balance. Studies have also confirmed the presence of stress-memory in M. galloprovincialis involving different responses at different times and eventual survival, such as from inflammation to control and resolve the inflammatory response and avoid subsequent cellular death and DNA damage after infection with Vibrio splendidus. The combination of a complex genome with the adaptation of the mussel immune system to a changing environment could explain this high variability [In model organisms such as shellfish, the inflammatory balance reaction ,57 is oniability ,43. The M. galloprovincialis, this signaling pathway forms a perfect balance with MAPK to control damage to the body. In contrast, the stress response of MAPK was relatively weak in A. irradians, and its balance regulation was self-limited, not via AMPK. We believe that this inflammatory balance system was closely related to the dominant physiological characteristics and immune advantages of M. galloprovincialis. However, A. irradians experienced higher mortality without such balanced regulation. In contrast, this unbalanced regulation simultaneously enables cells to overcome metabolic stress by inhibiting AMPK signaling [A. irradians. AMPK and PI3K-AKT are involved in the regulation of metabolites such as glucose. Therefore, we believe that the detoxifying system of A. irradians is more inclined to cellular metabolizing and apoptosis than toward inflammation regulation and self-protection.Novel insights into the specificity of AMPK signaling complexes have been gained . Studiesignaling , a key sM. galloprovincialis and A. irradians under natural algal bloom conditions. In order to explain the stress response difference between the two shellfish in the process of toxin metabolism, we divided the process into two metabolic stages. However, due to the long time span of the sampling, we could not accurately determine the activation time of AMPK, and thus further detailed research is needed. A. irradians exhibited high mortality, and although we were able to ensure that the experiments were conducted in sufficient numbers, A. irradians were taken alive and did not accurately reflect the condition of the large number of dead scallops. We mainly used the KEGG database for data analysis, which is strongly biased towards model organisms. The vast majority of bivalve genes only show limited sequence homology with sequences with a known function, which can often lead to incorrect annotations, and the signaling pathways in bivalves are not necessarily identical to those found in other organisms. In addition, autophagy and tissue damage are also common features in shellfish exposed to PSTs, although no relevant experiments were performed in this study.This is the first study to evaluate the differences in detoxification mechanisms for paralytic shellfish toxin in M. galloprovincialis and A. irradians under PSTs exposure. The significant changes found involved MAPK and AMPK signaling pathways and the specific regulatory pathway, PI3K-AKT. JNK and p38 MAPK activate the MAPK pathway in M. galloprovincialis. Meanwhile, there was significant expression of inhibitor genes, including TNF, NF-κB, and AMPK, especially during the slow metabolic phase. Therefore, an intense inflammatory reaction and a corresponding anti-inflammatory reaction in M. galloprovincialis constituted a relatively effective mechanism for high-filtering activity and low-tissue damage after PSTs exposure. However, A. irradians focused on the regulation of downstream cell metabolism and apoptosis, and thus the immune imbalance resulted in serious tissue damage and high mortality. In summary, there were clear differences in the metabolic patterns related to PSTs in M. galloprovincialis and A. irradians. Inflammation is an important factor that affects the metabolic pattern of PSTs in bivalves.An effective immune response is indispensable for growth, reproduction, and survival. In this study, we comprehensively compared transcription and physiological responses to examine the inflammatory–anti-inflammatory balance mechanisms in M. galloprovincialis (shell length: 46 mm ± 3.8 mm) and A. irradians (shell length: 65 ± 4.0 mm) were cultured in shell breeding cages under surface seawater (depth: 10 m) from January 2019. An Alexandrium catenella red tide was observed from 29 March until 13 May (<200 cells/L), with the highest density at 3.40 × 103 cells/L. From 2 April, sampling was performed every 3–6 days (depending on the sea conditions). Six biological replicates (PSTs accumulation × 3 and RNA-seq × 3) were collected each time, and each biological replicates contained 3–5 units. Three groups sampled on 11 April, 27 April, and 7 May were chosen for transcriptomics research. Shellfish hepatopancreas was collected on ice, washed with normal saline, and drained of surface water. All samples were immediately frozen in liquid nitrogen. Samples for sequencing were stored at −80 °C, whereas the samples for PSTs concentration measurements were stored at −20 °C until further analysis.The exposure experiment was conducted in the Qinhuangdao coastal culture area . One-year-old g for 5 min, and 1 mL of the supernatant was removed and adjusted for pH by 5 µL ammonium hydroxide, then centrifugation for 10 min at 16,000× g for purification of solid-phase extraction (SPE). ENVI-Carb cartridges  were activated with 3 mL acetonitrile, 3 mL 20% acetonitrile aqueous solution (containing 1% acetic acid), and 3 mL 0.1% ammonium hydroxide. After activation, 500 µL of the supernatant was added, and 700 µL ultrapure water was used for elution. Finally, 1 mL 75% acetonitrile solution (containing 0.25% formic acid) was used for elucidation. All samples were filtered through 0.22 μm syringe filters.Five grams of each sample was weighed into a 50 mL centrifuge tube after mixing with a homogenizer , and 5 mL of 1% acetic acid aqueous solution was added for extraction. After vortexing for 10 min, the mixture was heated using a boiling water bath for 5 min and allowed to cool to room temperature in flowing water. The samples were centrifuged at 10,000× LC-MS/MS analysis of the PSTs was performed using an HPLC system  coupled to a hybrid triple quadrupole linear ion trap mass spectrometer . PSTs were screened using the multiple reaction monitoring (MRM) method for the separation of toxins using a hydrophilic column  . The PSTThe hepatopancreas of shellfish were used for RNA extraction. RNA extraction and transcriptomic sequencing were performed by Gene Denovo, Guangzhou. The mRNA was enriched by Oligo (dT) beads. Sequencing was performed on an Illumina HiSeqTM 4000 platform. The paired-end sequencing with a 2 × 150 bp approach was applied. A total of 18 libraries were established . Clean data were produced by removing adaptor sequences, unknown nucleotides (>10%), and low-quality sequences  from the raw data for subsequent analyses. Then, transcriptome de novo assembly was carried out with the short reads assembling program Trinity. For each transcript, an FPKM value was calculated to quantify its expression abundance and variation using RSEM software. The unigene expression was calculated and normalized to RPKM (Reads Per kb per Million reads). Raw sequencing data has been uploaded to NCBI and the relevant accession numbers are PRJNA850029 and PRJNA850516.p-value) was calculated according to the model. Finally, multiple hypothesis tests were performed to correct the FDR values. We conducted two-by-two comparisons for the sampling groups 11 April, 27 April, and 7 May, respectively, where we set 11 April to 27 April as the Fast-stage and 27 April to 7 May as the Slow-stage. Based on the results of analysis of variance, genes with p-value < 0.05 and |log2FC| > 1 were screened as significantly different genes. GOseq software was used to annotate the GO functions in order to reveal the biological functions of the differentially expressed genes; KEGG was used to analyze the signal pathways involving the genes. A threshold-corrected p-value < 0.05 was used to determine the significance of differences in GO and KEGG annotations of DEGs. Moreover, we performed gene set enrichment analysis using the software GSEA and MSigDB to identify whether a set of genes in a specific KEGG pathway showed significant differences between the two groups. Significantly enriched gene sets were defined as those with normalized enrichment score (NES) > 1 and p-value < 0.05.Differential expression analysis was performed using DESeq2 software. The read count was first normalized, and then the probability of hypothesis testing .All data were analyzed using SPSS version 16.0 for Windows . Two-tailed Student’s


E and network pharmacology analysis were performed to explore active ingredients and therapeutic mechanisms of TXS. TXS treatment decreased levels of immunoglobulin E (IgE), interleukin-4 (IL-4), and tumor necrosis factor-α (TNF-α) in serum induced by DNCB. TXS reduced scratching behavior and alleviated inflammatory pathology of skin and ear. Meanwhile, TXS decreased the spleen index and increased spleen index. The UPLC-Q-Exactive-MSE results showed that 65 compounds of TXS were detected and 337 targets were fished. We collected 1371 AD disease targets, and the compound-target gene network reveled that the top 3 active ingredients were (−)-epigallocatechin gallate, apigenin, and esculetin, and the core target genes were PTGS2, PTGS1, and HSP90AA1. The KEGG pathway and GO analysis showed that TXS remedied atopic dermatitis via PI3K-Akt signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and Toll-like receptor (TLR) signaling pathway with the regulation of inflammatory response and transcription. Further, we found that the targets of PTGS2 and HSP90AA1 were both elevated in ears and skin of AD model mouse; however, TXS decreased the elevated expressions of PTGS2 and HSP90AA1. Our study revealed that TXS ameliorated AD based on (−)-epigallocatechin gallate, apigenin, and esculetin via targeting PTGS2 and HSP90AA1.Atopic dermatitis (AD) is a relapsing and chronic skin inflammation with a common incidence worldwide. Ta-Xi-San (TXS) is a Chinese herbal formula usually used for atopic dermatitis in clinic; however, its active compounds and mechanisms of action are still unclear. Our study was designed to reveal the pharmacological activities, the active compounds, and the pharmacological mechanisms of TXS for atopic dermatitis. Mice were induced by 2,4-dinitrocluorobenzene (DNCB) to build atopic dermatitis model. The pathological evaluation, enzyme-linked immunosorbent assay (ELISA), and hematoxylin and eosin (H&E) assay were performed. The UPLC-Q-Exactive-MS It is ond IFN-γ , 9. Morend IFN-γ , 11.Sophora flavescens Aiton , Phellodendron amurense Rupr. , Sanguisorba officinalis L. Bunge , Portulaca oleracea L. , Atractylodes lancea (Thunb.) DC. , and Alumen , which has widely used in the Shaanxi Traditional Chinese Medicine Hospital to treat skin swelling, itching, papules, and other dermatitis symptoms for nearly 15 years. The traditional uses of TXS consist of reducing heat and expelling toxins, expelling pus and dispersing swelling, and cooling the blood and hemostasis, all of which are associated with atopic dermatitis [Sanguisorba offcinalis L. has the ability to promote wound healing, relieve pains, and reduce inflammation reactions and tissue edema [Phellodendron amurense protects human keratinocytes from PM2.5-induced inflammation and contains the bioactive compounds for atopic dermatitis management [Phellodendron amurense can reduce the skin swelling and ear tissue weight of the AD mouse induced by 2,4-dinitrochlorobenzene [Sophora flavescens presents effects of anti-inflammation and antioxidant [Sophora flavescens was used for AD through increasing the IL-12 and IFN-γ mRNA levels and reducing the ICAM-1, MDC, and SOCS1 mRNA levels [κB signaling pathway [α, IL-6, and IL-1β production in lipopolysaccharide-induced acute lung jury [Phellodendron amurense, suppressed the release of IL-6, CXCL8, and chemotactic factors in the eosinophils-dermal fibroblasts coculture [Ta-Xi-San (TXS) is a Chinese herbal formula, consisting of six herbs: rmatitis –15. In tue edema . The hernagement , 17. Theobenzene . In addiioxidant , 20. MorA levels . Gallic  pathway . Atractyung jury , 24. Beroculture . HoweverE and network pharmacology study to illuminate potential mechanism of TXS.Network pharmacology has made a significant contribution to systematically reveal the function and behavior between herbal formulas . This reα, and IgE ELISA kits were obtained from Nanjing Keygen Biotech . PTGS2 and HSP90AA1 ELISA kits were purchased from R&D .TXS was obtained from Shaanxi Academy of Traditional Chinese Medicine. Methanol and formic acid (LC-MS-grade) were purchased from Thermo Fisher Scientific Co., Ltd. . 2,4-Dinitrofluorobenzene (DNCB) was purchased from Macklin Biochemical Technology Co., Ltd. Hydrocortisone Butyrate Cream was obtained from Hunan WZT Pharmaceutical Co., Ltd. . The rat IL-4, TNF-TXS  was soaked with 1000 mL in water for 30 min, decocted for 5 min again after boiled, and then filtrated. The decoction filtrated was concentrated to 160 mL (1 g crude drug/mL). The solution was filtered using 0.22 mm membranes. The filtrate was used and stored at 4°C before its use for animal assays.n = 6 per group): (1) normal group, (2) matrix group, (3) DNCB model group, (4) DNCB + matrix + 0.65 mg crude drug/mL TXS, (5) DNCB + matrix + 1.30 mg crude drug/mL TXS, and (6) Hydrocortisone Butyrate Cream (Hyd B). Firstly, the mice were shaved with an aesthetic state (ethyl carbamate) using an electric shaver. Then, 100 μL 0.5% DNCB or acetone/olive oil (3 : 1) was applied to the shaved dorsal skin for 2 days, and there was no treatment in the 3rd∼6th days . 70 μL 0.5% DNCB on dorsal skin and 20 μL 0.5% DNCB on ear every 2 days were applied from the 7th day. The AD model was finished on the 28th day for a total of 10 times . DNCB was applied on the same site r. TXS was applied from the 7th day, 500 μL on dorsal skin and 200 μL on ears, twice a day until the 28th day. Hyd B was also applied from the 7th day, 300 mg on dorsal skin and 70 mg on ears, twice a day until the 28th day. The flowchart of the experimental program we studied is shown in BALB/c mice  were purchased from Xi'an Jiao Tong University . All the mice were fed under the standard experimental conditions . The AD mice model was induced by DNCB (dissolved in acetone/olive oil (3 : 1)), which was applied to dorsal skin and ear. Mice were segmented into 6 groups  eryThe spleen and thymus of mice in each group were removed integrally. The thymus index and spleen index were defined as the organ/body ratio (organ mass (mg)/corresponding animal mass (g)). The organ and the body weight were calculated using an electronic balance.μm thick, and then they were stained with H&E.The dorsal skin and ear samples were taken 24 h after final herbal administration and then fixed in 4% paraformaldehyde buffer, followed by embedding of the tissues in paraffin wax. The tissue sections were 2 α and IL-4, were also quantified using a mouse cytokine enzyme immunoassay kit, respectively.Total IgE in serum was analyzed using an ELISA Quantitation Kit according to the manufacturer's protocol. The serum concentrations of the cytokines, including TNF-μm). The mass spectrometer used in the UPLC-Q-Exactive-MSE analysis was a quadrupole-electrostatic field orbital with an ESI mode and data treating used Mass Lynx 4.1 software. Column temperature was 30°C. The mobile phase consisted of 2 solvents: (A) 0.1% formic acid aqueous solution and (B) methyl alcohol . The flow rate was kept at 0.3 mL/min. The injection volume was set as 1 μL. The ion source of thermoelectric spray (HESI) MS was performed to collect data in full switch ion mode. Other parameters were as follows: capillary temperature: 300°C, auxiliary gas heater: 300°C, sheath gas flow rate: 30 L/min, spray voltage: 3.50 kV, full MS resolution: 7000, ddms2 resolution: 175000, and scan range: 100 to 1 200 Da. S-lens RF level was set as 55 V.The TXS aqueous extract was analyzed with a 150 mm × 2.1 mm Accucore AQ C18 column  and the Comparative Toxicogenomics Database  based on UPLC-Q-Exactive-MSE analysis. The UniProt (https://www.uniprot.org/) database was used for drug target revision to match the official name. The Online Mendelian Inheritance in Man , Comparative Toxicogenomics Database  and GeneCards-Human Genes (GeneGards: https://www.genecards.org/) database were used to collect the AD-related disease genes.In this work, we search the targets of UPLC-Q-Exactive-MSFor better dissecting the latent active ingredients and key targets of TXS, we established the herb-ingredient-target-pathway network using Cytoscape 3.7.1 software. Then, the quantitative “degree” was analyzed by plugin network analyzer. According to the descending order of “degree,” we selected the vital ingredients and targets of TXS against AD.https://www.genome.jp/kegg/pathway.html) assay was performed based on the clusterProfiler software package on R platform and analyzed using visualization. Afterwards, the GO interactive network and the bubble diagram of KEGG pathway were structured using the top GO packet of R platform. P < 0.05 was calculated meaningfully in these two enrichment analyses.The Gene Ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway .t-test. P < 0.05 indicated significant difference between groups.Our data are shown as the mean ± standard deviation (SD). Statistical comparison among groups was analyzed with GraphPad Prism 5.01 . ANOVA was firstly used followed by Student's The skin inflammation of the back and ear of mice in each group was observed by visual method . The skiAt the same time, the lesion of back skin was scored according to the “inflammatory score standard.” Compared with the normal mice, the back inflammatory score of mice in DNCB group was significantly enhanced, and TXS (1.3 mg crude drug/mL) significantly reduced the inflammatory score . There wAs Figures Hematoxylin study was used to recognize whether TXS regulates pathological symptoms of AD-like skin lesions and ear injury in DNFB-induced AD mouse model. In model group, skin tissues of mice showed typical inflammatory pathological changes including parakeratosis, thickening of the spinous layer, telangiectasia, and infiltration of inflammatory cells in the dermis . In miceα, and IgE in serum were significantly increased in the model group's mice induced by DNFB compared to the normal group. However, production of IL-4, TNF-α, and IgE was inhibited by TXS and Hyd B treatment  of TXS extracts in positive ion mode and negative ion mode is shown in Figures The herb components of TXS extracts were detected via aligning each detectable compound mass data for each detectable compound with structure and confirmed by MSE analysis after eliminating the duplicate targets. Simultaneously, 1371 AD-related targets were collected via searching CTD, GeneCards, and OMIM databases, and 1241 targets were decontaminated after removing duplicates. Then the compound targets and disease-related targets were merged, and we got the compound-target gene network.The 337 targets were collected form the 65 compounds in TXS based on UPLC-Q-Exactive-MSThe ingredient-target gene network is presented in KEGG pathway enrichment showed that 199 pathways were harvested and the remarkable 30 related pathways are presented in PTGS2 and HSP90AA1 are major regulatory targets for inflammatory response in AD and are core targets screened via network pharmacology. Thus, we investigate the regulatory effect of TXS on activation of PTGS2 and HSP90AA1. In our study, we found that the protein expressions of PTGS2 and HSP90AA1 in ears and skin of model mice were significantly enhanced compared to the normal mice; however, TXS decreased the elevated expressions of PTGS2 and HSP90AA1 Figures . Our resα are risk factors for developing atopic dermatitis [α, and IL-1β-were enhanced in serum of AD patients [γ and IL-6 was detected in skin lesions of AD patients [α, INF-γ, and IgE in serum were reduced; meanwhile, the inflammation of back skin, the skin inflammation score, and the ear swelling of mice were alleviated after taking the TXS. Hence, TXS might be effective to balance atopic and inflammatory disorders in the progress of atopic dermatitis.The DNCB-induced skin lesion in mice is a classical animal model for AD research . Thus, wrmatitis . Previoupatients . Higher patients . In our In addition, to explore the effects of TXS on skin and ear inflammation, we performed H&E assay and examined thymus index and spleen index. The mice induced by DNFB showed typical AD features, such as epidermal hyperplasia and massive inflammatory cells infiltration, which were coincided with the typical symptoms of AD. TXS treatment effectively improved the skin and ear status. The thymus is a major immune organ, the index of which is an important index for assessing immunity . MeanwhiE as a rapid, efficient, and sensitive method is always used for analyzing extracts and bioactive ingredients from medicine and herbs [E to analyze the information on compounds in herbal medicines of TXS. 65 components in TXS were identified, including alkaloids, phenols, flavonoids, and acids. Among them, various ingredients showed anti-inflammatory activity. Matrine, a quinolizidine alkaloid extracted from Sophora flavescens, has the capacity to effectively suppress experimental autoimmune encephalomyelitis and optic neuritis [garden burnet, epiberberine from golden cypress, and atractylenolide I from Rhizoma atractylodis showed anti-inflammatory activity [UPLC-Q-Exactive-MSnd herbs , 38. Theneuritis , 40. Oxyneuritis , 42. Epiactivity –45. Thusα, IL-5, and IL-13) production to decrease allergic responses in RBL-2H3 cells and repairs the physical barrier of the skin in HaCaT cell model [γ, IL-13, IL-31, and IL-17 release via regulating nuclear factor-κB pathway [The network pharmacology study as a major tool systematically revealed the effects and mechanism of multicomponent and multitarget system, such as traditional Chinese medicine , 47. Herll model . Esculet pathway . FurtherSolanum nigrum Linne possessed anti-inflammatory effects in vitro through regulating MAPK/NF-κB pathway in HaCaT cell model [Artemisia argyi Folium extract ameliorates DNCB-induced skin lesions in BALB/c mice via inhibiting PI3K/Akt pathway [Based on the enrichment results of KEGG, we determined that the effects of TSX against AD may be due to pathways like PI3K-Akt signaling pathway, MAPK signaling pathway, Th17 cell differentiation, and TLR signaling pathway. The mitogen-activated protein kinase (MAPK) pathway is responsible for inflammatory and immune response and is widely expressed in multiple tissues . Previoull model . It has ll model . Artemis pathway . In thisIn conclusion, our results revealed that TXS attenuated IL-4, IgE, and TNF-ɑ levels in serum, increased thymus index, and decreased spleen index. TXS ameliorated behavioral changes and alleviated pathological changes of skin and ear in mice with the DNCB-induced atopic dermatitis. Additionally, TXS was mainly consisted of 65 chemical components. Among them, 52 components were closely associated with the effect of TXS against AD. TXS downregulated PTGS2 and HSP90AA1, which were associated with the PI3K-Akt signaling pathway. Our results revealed the effects, components, and pharmacological mechanism of TXS for AD, which provided scientific basis for clinical application.


Background: This study was performed to identify key regulatory network biomarkers including transcription factors (TFs), miRNAs and lncRNAs that may affect the oncogenesis of EBV positive PTCL-U.Methods: GSE34143 dataset was downloaded and analyzed to identify differentially expressed genes (DEGs) between EBV positive PTCL-U and normal samples. Gene ontology and pathway enrichment analyses were performed to illustrate the potential function of the DEGs. Then, key regulators including TFs, miRNAs and lncRNAs involved in EBV positive PTCL-U were identified by constructing TF–mRNA, lncRNA–miRNA–mRNA, and EBV encoded miRNA–mRNA regulatory networks.Results: A total of 96 DEGs were identified between EBV positive PTCL-U and normal tissues, which were related to immune responses, B cell receptor signaling pathway, chemokine activity. Pathway analysis indicated that the DEGs were mainly enriched in cytokine-cytokine receptor interaction and chemokine signaling pathway. Based on the TF network, hub TFs were identified regulate the target DEGs. Afterwards, a ceRNA network was constructed, in which miR-181(a/b/c/d) and lncRNA LINC01744 were found. According to the EBV-related miRNA regulatory network, CXCL10 and CXCL11 were found to be regulated by EBV-miR-BART1-3p and EBV-miR-BHRF1-3, respectively. By integrating the three networks, some key regulators were found and may serve as potential network biomarkers in the regulation of EBV positive PTCL-U.Conclusion: The network-based approach of the present study identified potential biomarkers including transcription factors, miRNAs, lncRNAs and EBV-related miRNAs involved in EBV positive PTCL-U, assisting us in understanding the molecular mechanisms that underlie the carcinogenesis and progression of EBV positive PTCL-U. Peripheral T cell lymphoma, unspecified (PTCL−U) is one of the most common subtypes of mature post-thymic T cell neoplasms, which consists of variable immunophenotypes and morphological changes . PTCL-U Gene expression regulation is one of the key mechanisms for controlling biological processes in the organism . TranscrRecently, bioinformatics analysis as an interdisciplinary field has been regarded as a reliable technique for revealing molecular markers and therapeutic targets that may be implicated in the pathogenesis of cancer or other diseases . In the In this study, some key regulators including TFs, miRNAs and lncRNAs involved in EBV positive PTCL-U were identified and integrated them into a strong theoretical framework through constructing TF–mRNA, lncRNA–miRNA–mRNA, and EBV encoded miRNA–mRNA regulatory networks. The present study may offer new insights into the molecular mechanism of EBV positive PTCL-U and provide potential network biomarkers for EBV positive PTCL-U. The flowchart diagram for this study is represented in The microarray data for GSE34143 in the GEO database were obtained on the GPL6480 and GPL14550 platform. The expression profiles are provided for nine samples, including 3 EBV positive PTCL-U and 6 normal reactive lymph nodes.p-value < 0.05 were considered statistically significant. The analysis results were presented by volcano map.GEO2R is an interactive web tool that allows users to compare two or more sets of samples in the GEO Series and contributes to filtrating DEGs through the Bioconductor’s GEO Query and Limma R software . We usedp-value < 0.05 was considered statistically significant.To further evaluate the function of the DEGs, KEGG pathway and GO enrichment analyses were conducted. The GO functional enrichment analysis was applied to annotate genes and gene products, and also accomplish characteristic biological function originating from genomic or transcript omic data at three different levels including biological process (BP), molecular function (MF), and cellular component (CC) . KEGG haSearch Tool for the Retrieval of Interacting Genes (STRING) is a database resource that can investigate complex PPI information . We retrNetwork analysis was carried out to identify the key hub mRNAs with high degrees in the initial network. Module analysis was applied to screen the significant modules active in the network with the Molecular Complex Detection (MCODE) plugin in Cytoscape . Then, whttp://www.rna-society.org/virbase) is an online tool that provide a resource for efficient browsing and visualization of interaction networks in virus-host non-coding RNAs (ncRNAs) associated interactions  PTCL with normal/reactive tissue is not going to reveal the significance of EBV but that of the neoplastic process itself. Second, the sample size for the rare disease is too small while investigating a larger sample size in EBV positive PTCL-U would help to confirm our data. Third, the data in the current study were identified by bioinformatics analysis, and the findings are warranted to be validated by further investigations. Thus, further biological experiments are needed to explore the biomarker value of the identified network regulators and the underlying mechanisms. Last, we should also pay attention to the quality control of the assay and data generation letter analysis process, and the combination of NGS, GEP, CNA and other multi-omics analysis, which is beneficial to the molecular typing of PTCL-U and the search for new therapeutic targets.In summary, a network-based approach to identify potential biomarkers involved in EBV positive PTCL-U was developed by integrating the regulatory networks, including TF-mRNA, lncRNA-miRNA-mRNA, EBV-encoded miRNA-mRNA. Two transcription factors, including PAX5 and SPIB, were identified that were both associated with the tumorigenesis of EBV positive PTCL-U through regulating their target genes. Four hub mRNAs in this network, including CXCL10, CXCL11, CCL7, and CCL8, were identified which have also been demonstrated to influence the tumor microenvironment and infection status. Four miRNAs  and one unreported lncRNA, LINC01744, were identified in the ceRNA regulation network. In addition, EBV related miRNAs EBV-miR-BART1-3p and EBV-miR-BHRF1-3 were found to regulate CXCL10 and CXCL11 in the network. This study may provide a new avenue for investigating the regulatory mechanisms of EBV positive PTCL-U, and further molecular experiments are required to confirm these findings.


Purpose: Modern, open-source databases provide an unprecedented wealth of information to help drug development. By combining data available in these databases with the proper bioinformatical tools, we can elucidate the molecular targets of natural compounds. One such molecule is curcumol, a guaiane-type sesquiterpenoid hemiketal isolated from Rhizoma Curcumae, which is used for a broad range of diseases in traditional Chinese and Indian medicine. It has been reported to exert anti-tumor activity, but the intrinsic molecular mechanism in hepatocellular carcinoma (HCC) is unclear. Therefore, the present study was designed to reveal the predictive targets and biological mechanisms of curcumol against HCC via a network pharmacology-based approach combined with bioinformatic analytics and to provide proof of concept for further similar investigations. Methods: Data available from open-source databases  was processed with the help of a variety of open-source tools . Results: In the present study, the potential of curcumol against HCC was unraveled by network pharmacology-based elucidation. It suggests that curcumol shows exciting druggability with 44 potent homo sapiens biotargets against HCC. The GO terms and KEGG pathways enrichment analyses, curcumol-targets-pathways-HCC network, PPI network, and corresponding in-depth topological analyses, as well as survival analysis, molecular docking simulation indicate that the potential mechanism of curcumol against HCC is complicated, as it may act in various ways, mainly by inducing apoptosis and modulating the inflammatory response, increasing presentation of HCC-specific protein. Conclusion: The present study highlights the potential of curcumol against HCC, giving reference to further experimental study. It also presents a roadmap that can be followed to conduct in silico prescreening of other compounds of interest. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer that occurs most often in people with long-term liver diseases, especially cirrhosis caused by hepatitis viral infection .Natural products from traditional herbal medicines are precious sources for new drug discovery. Curcumol, as one of the typical examples, is a bioactive extract of essential volatile oils from Curcumae Rhizoma , which hWe first conducted an “absorption, distribution, metabolism, and excretion” (ADME) evaluation by using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and SwissADME analysis platform to investigate the druggability of curcumol at the molecular level.Then, putative targets of curcumol were mined from Comparative Toxicogenomic Database (CTD) and SwissTargetPrediction server. Meanwhile, we obtained the therapeutic targets of HCC from The Cancer Genome Atlas (TCGA) cohort and Genotype-Tissue Expression (GTEx) portal, GeneCards database, and Online Mendelian Inheritance in Man (OMIM) database. An intersection containing all the common targets of the putative targets of curcumol and the therapeutic targets of HCC was calculated by the JVenn program.Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses were carried out by WEB-based GEne SeT AnaLysis Toolkit  for these common targets to gain a macroscopic understanding of their functions.Moreover, a preliminary protein-protein interaction (PPI) network was constructed by the GeneMANIA plug-in of Cytoscape software. To have a deeper insight into the network, a cluster classification of the preliminary PPI network was performed by the MCODE plug-in of Cytoscape software. Each cluster was followed by GO terms and KEGG pathways analyses. In addition, by using the CytoNCA plug-in of Cytoscape software, a core PPI subnetwork with 9 key nodes and 22 edges was extracted.Moreover, a survival analysis was also performed for the common targets, where we found 17 significantly prognosis-related genes from various types of survival.By using the JVenn program again, we sorted out two key targets  from the core PPI subnetwork that are significantly prognosis related. We then, from the aspects of cancer grading and Spearman correlation analysis, provided a brief summary of RELA and SQSTM1 to show their importance in HCC. Furthermore, with the help of the Human Protein Atlas project, we clinically verified their importance in HCC patients by comparing the expression in normal liver tissue and pathological liver tissue. Consequently, it was found that they themselves are positively correlated, and their overexpression is associated with HCC carcinogenesis.Finally, molecular docking and molecular dynamic simulation were performed to investigate their binding to the curcumol molecule.http://www.swissadme.ch/, accessed on 22 November 2021) is an online tool that offers structure based ADME parameter prediction. In the present study, given the three-dimensional structure of the curcumol molecule, it was first used to roughly assess its druggability. Then, we searched with the keyword “Curcumol” on the TCMSP database  to further confirm the pharmacological properties in exact values in a literature-based manner [SwissADME server  is a publicly available digital ecosystem that relates toxicological information on chemicals, genes, phenotypes, and diseases, to literature data [The Comparative Toxicogenomic Database  is a structure-based target predictor [https://pubchem.ncbi.nlm.nih.gov/, accessed on 22 November 2021) [SwissTargetPrediction  ,15 and ihttps://portal.gdc.cancer.gov/, accessed on 22 November 2021) [https://gtexportal.org/home/, accessed on 22 November 2021) [https://www.aclbi.com, accessed on 22 November 2021) for statistical computing [https://www.genecards.org/, accessed on 22 November 2021) [https://www.omim.org/, accessed on 22 November 2021) [Raw counts of RNA-sequencing data (level 3) and corresponding clinical information were obtained from the TCGA cohort er 2021) . Normal er 2021)  as part omputing . In addier 2021)  and OMIM21 . Normhttp://jvenn.toulouse.inra.fr/app/index.html, accessed on 22 November 2021) is a JavaScript-based program that can exert a Venn diagram of up to 6 data sets [http://bioinfo.life.hust.edu.cn/GSCA/, accessed on 22 November 2021) [The JVenn program  [http://www.bioinformatics.com.cn/en, accessed on 22 November 2021). Consequently, by using Cytoscape software, we constructed a curcumol-targets-HCC-pathways network [To gain a macroscopic understanding of the functions of common targets, we carried out GO terms and KEGG pathway enrichment analyses by WebGestalt (er 2021) . The res network .The GeneMANIA plug-in of Cytoscape software is a robust application that can analyze and prioritize functional assays . Given qAfter obtaining the preliminary PPI network, MCODE, a Cytoscape plug-in, was used to cluster the protein network . The parCytoNCA, another Cytoscape plug-in that is capable of network centrality analysis, was used to extract the core PPI subnetwork via in-depth topological analyses . We firsp values and hazard ratio (HR) with a 95% confidence interval (CI) were generated by log-rank tests and univariate COX proportional hazards regression. The results were plotted as forest plots and Kaplan–Meier (KM) curves.To identify the targets that were prognosis related in HCC, a total of 44 common targets underwent survival analysis, including overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and disease-specific survival (DSS) through R packages “ggplot2” and “survival” on Scihub ,29. The By using JVenn program to calculate the intersection of the 9 key nodes from step 4.3 and the 17 significantly prognosis-related genes from step 5, 2 key targets  were found.p < 0.05 was considered statistically significant [In order to unravel the importance of the key targets, we conducted Kruskal–Wallis test to compare their expression levels in various cancer grades  and normal tissues. Afterward, we performed a receiver operative characteristic (ROC) analysis to see if RELA and SQSTM1 are eligible to be the biomarker in HCC. Tumoral RNA-seq data were downloaded from the TCGA cohort, while data of normal tissue samples were from the GTEx portal. We also used the Spearman method to describe the correlation between RELA and SQSTM1. All the statistical analyses were performed by R packages “ggstatsplot” and “pROC” on SciHub, where nificant ,31.https://www.proteinatlas.org/, 26 November 2021), we inspected the histological slides of HCC patients and normal individuals [https://www.proteinatlas.org/ENSG00000173039-RELA 26 November 2021 and https://www.proteinatlas.org/ENSG00000161011-SQSTM1 26 November 2021.At this step, by using the Human Protein Atlas project  were acquired and prepared in PyMOL (v1.2r3pre) by removing water molecules, adding charge, and parameterizing for molecular docking on AutoDock Vina-based platform, CBdock  [https://alphafold.ebi.ac.uk/entry/Q13501, 28 November 2021) for docking [To simulate the interaction of the key targets with the curcumol molecule, structures of the key targets from PDB  ,34,35,36 docking ,38.Molecular dynamics simulations were performed to investigate the binding conformational stability of the protein-ligand complex. The stability of the protein-ligand complex was maintained during 100 ns simulations using Desmond software for compounds based on root mean square deviation (RMSD), root mean square fluctuation (RMSF), and hydrogen bond interactions ,40.2 usually means the molecule is poor at penetrating the cell membrane, while if TPSA < 60 Å2, usually it is suitable at penetrating the cell membrane.During ADME evaluation, multiple pharmacological parameters are discussed . MW (molecular weight) refers to the mass of a molecule, Hdon and Hacc refer to hydrogen bond donor and receptor, AlogP value represents the partition coefficient between octanol and water . They ar2. From this aspect, as the curcumol molecule possesses the following parameters: OB = 103.55%, DL = 0.13, TPSA = 0.25 Å2, RBN = 1, it’s deemed to possess exciting druggability , progression-free survival (PFS), disease-free survival (DFS), and disease-specific survival (DSS). In total, 17 genes were identified as prognosis related in HCC in case of different types of survival.By considering p values, hazard ratios (HR) with 95% confidence interval (IC) in different types of survival . In summary, through the survival analysis, except GPT, PGR, and HSD17B2, the rest  are related to poor prognosis.Their corresponding KM curves are available in p-value < 0.001. This hints that they have potential synergistic effects.By using the JVenn program, two key targets, RELA and SQSTM1, were sorted out from the intersection of the nine key nodes and the significantly prognosis-related genes, as shown in Moreover, we also explored their expression levels at different HCC grades and normal tissues by Kruskal–Wallis’s test. The result is shown in In order to verify whether RELA and SQSTM1 may serve as reliable biomarkers in HCC, we further performed a diagnostic receiver operative characteristic (ROC) analysis, as shown in In order to verify the importance of RELA and SQSTM1 proteins in HCC patients, we compared their expression with that of normal individuals. Both proteins showed relatively low expression in normal tissues, while in the histological slides from HCC patients, they exhibited high expression levels, as shown in We downloaded the corresponding three-dimensional structures from RCSB PDB to conduct blind docking. The docking site was set in a cubic box in the center of the initial ligand. The results of RELA docking indicates that its binding with curcumol possesses two hydrogen bonds with ARG 187 and ALA 188 and two hydrophobic interactions with ASP 277 and ASN 190, while the results of SQSTM1 docking indicates that its binding with curcumol possesses two hydrogen bonds with ARG 187 and ALA 188 and two hydrophobic interactions with ASP 277 and ASN 190.Moreover, as the more negative the docking score is, the higher the binding force is between the compound and the protein, we, therefore, found the best run provided the binding affinity of −5.7 kcal/mol and −5 kcal/mol to RELA and SQSTM1, respectively, taking together with their specific interactions into account. As generally speaking, affinity energy ≤ −5 kcal/mol is deemed as high affinity. RELA and SQSTM1 thus can be considered as key players that participate in the potential mechanism against HCC of curcumol. The docking results can be viewed in a three-dimensional manner in Molecular dynamics simulations were performed on the top hits containing high binding energies. Over the simulation period, the projected conformational changes from the initial structure were presented in RMSD. Moreover, structural stability, atomic mobility, and residue flexibility at times of interaction of protein-hit were expressed with RMSF values. The peaks of the RMSF graph represent the fluctuation portion of the protein through the simulation. The N- and C-terminals show more changes than any other portion of the protein. α helices and β strands show less fluctuation, as they are stiffer than the unstructured part of the protein than the loop portion. The RMSD of the curcumol-RELA complex showed deviation at almost 30 ns for 2 Å, and then the system was equilibrated throughout the simulation, as shown in The curcumol-RELA complex possessed several different types of interactions, including hydrogen bonds, water bridges, and hydrophobic, as shown in The broad use of essential volatile oils from Curcumae Rhizoma in the treatment of many diseases, including tumors, has a long and storied history. While HCC is a fatal major malignance among liver cancers, previous studies have not yet explored the potential of curcumol against this form of the disease. Therefore, the present study attempted to make the first step to address this issue. As blindly conducting experiments on a large scale is both time and resource consuming, in silico method was employed in the present study in order to improve the effectiveness and save labor.In the present study, out of 371 HCC samples and 276 normal tissue samples, 44 common targets were mined from various authorized databases . With these targets, functional analyses were carried out to gain a macroscopic understanding of their roles against HCC. It appears that curcumol could function by inducing apoptosis and inflammation, disturbing vessel generation of the tumor. Notably, VEGFA appears to be down-regulated so that vascularization of tumor tissue can be suppressed. These findings suggest that curcumol may function against HCC via inducing cell death and immunological reactions, as well as disturbing intra-tumor vascularization  and elimMoreover, a preliminary PPI network was also constructed, trying to gain in-depth insight into their functions. Via topological optimization, we conducted functional clustering for the preliminary PPI network. Each cluster was followed by GO terms and KEGG pathways enrichment analyses through which we found that proteins in cluster 1 were mainly associated with apoptosis, whereas proteins in cluster 2 were mainly associated with neurological signaling. Moreover, a core PPI subnetwork with nine key nodes was extracted, which was used to screen key targets in the following analyses.In addition, we conducted a survival analysis for different types of survival  for the 44 common targets where 17 genes were considered as significantly prognosis related.Among them, 2 key targets, SQSTM1 and RELA, were sorted out by intersecting between the 9 key targets and these 17 significantly prognosis-related genes. From the aspects of cancer grading and Spearman correlation analysis, we provided a brief summary of RELA and SQSTM1, which emphasized their importance in HCC with ROC curve plotted to evaluate the reliability of our analyses. Then, we conducted in situ analysis by using the histological slides from HCC tissues and normal tissues. It was found that both RELA and SQSTM1 showed relatively low expression in normal tissues, while in the histological slides from HCC patients, they exhibited high expression levels, indicating that our computational prediction is in suitable agreement with the observation results. Consequently, it was found that their overexpression is statistically significant in tumor tissues, and they themselves are correlated with poor prognosis.Via molecular docking, we found RELA and SQSTM1 showed high affinity to the curcumol molecule and are thus thought to be the key players in the potential anti-HCC mechanism of curcumol. Therefore, we further verified the stability of their binding by performing molecular dynamic simulation to ensure the binding was not affected by different docking protocols. As a result, we found that both the RELA-curcumol complex and SQSTM1-curcumol complex were stable during 100 ns-long molecular dynamic simulations. Combined with current literature, it is believed that curcumol decreases the expression level of RELA  and has Although further experiments are needed, the present study elucidated the potential of curcumol against HCC, providing an important basis for in vivo studies. The approach described in the current work could also be used to screen for other biologically active compounds used in traditional Eastern medicine.In summary, the present study demonstrated the potential underlying mechanism of curcumol in HCC treatment based on a network pharmacology method. We revealed that curcumol seemed to play a critical role in HCC by affecting multiple biotargets. Moreover, our molecular docking and molecular dynamic simulation results also suggested that the RELA and SQSTM1 proteins could bind with curcumol molecule stably, providing an important basis for further investigation, although this study also has certain limitations as pharmacological and clinical research still need to further validate our findings.