The database source code and content can be downloaded through GitHub (https://github.com/bic-nirrh/precocity).Precocious puberty (PP) is an important endocrine disorder affecting children globally. Several genes, SNPs and comorbidities are reported to be associated with PP; however, this data is scattered across scientific literature and has not been systematically collated and analysed. In this study, we present PrecocityDB as the first manually curated online database on genes and their ontology terms, SNPs, and pathways associated with PP. A tool for visualizing SNP coordinates and allelic variation on each chromosome, for genes associated with PP is also incorporated in PrecocityDB. Pathway enrichment analysis of PP-associated genes revealed that endocrine and cancer-related pathways are highly enriched. Disease enrichment analysis indicated that individuals with PP seem to be highly likely to suffer from reproductive and metabolic disorders such as PCOS, hypogonadism, and insulin resistance. PrecocityDB is a useful resource for identification of comorbid conditions and disease risks due to shared genes in PP. PrecocityDB is freely accessible at  Disease-centric databases significantly contribute to advancement of health research as they offer curated and well-annotated data amenable for further analysis. While many of the diseases such as cancers, autism, Alzheimers, PCOS and endometriosis have dedicated databases, it would be worthwhile to include more diseases in this list. In the present study, we have reviewed scientific literature on precocious puberty (PP) and developed a manually curated database with information on genes associated with PP, their ontology terms, SNPs and pathways.1. PP is statistically defined as early pubertal development at an age that is 2.5 standard deviations lower than the mean age of pubertal onset2.Puberty is characterized by growth and maturation of primary sexual organs leading to development of secondary sexual characteristics. Puberty attained before age of 8 years in girls and 9 years in boys is considered as precocious4. Activation of the HPG axis, nearing the pubertal age, stimulates the anterior pituitary to secrete gonadotropins such as FSH and LH, in response to which gonads develop. This process is termed as gonadarche. Gonadarche is accompanied by increased secretion of gonadal sex steroid hormones such as estrogen and testosterone. Prior to and independent of gonadarche, adrenarche occurs when there is a surge in the secretion of adrenal androgens such as DHEA and DHEA-S5 leading to growth and appearance of sexual hair8.The onset of puberty is initiated by the maturation of hypothalamic–pituitary–gonadal (HPG) axis that ultimately leads to sexual dimorphism and fertility9. The overall prevalence of PP is in the range of 1: 5000 to 1: 10,000 children10. CPP is more common in girls with a female to male ratio that ranges from 3:1 to 23:1. Some rare cases of CPP triggered due to organic causes are more common among boys12. IPP comprises of variants of pubertal development like premature adrenarche, premature thelarche, and premature menarche that may lead to PP7.PP can be sub-classified as (i) gonadotropin dependent or central precocious puberty (CPP); (ii) gonadotropin independent or peripheral precocious puberty (PPP); and (iii) incomplete precocious puberty (IPP). CPP is caused due to premature activation of HPG axis whereas PPP is associated with increased gonadal secretion of steroidal sex hormones7.PP is diagnosed based on physical and biochemical changes that are associated with puberty. Assessment is done using detailed family history, bone X-ray, brain MRI, and hormonal profiling10. Early onset of puberty profoundly impacts the psychosocial well-being of individuals14. Children with early puberty experience higher levels of behavioural and psychological disturbances as compared to children with normal puberty. Individuals with PP suffer from short stature due to premature fusion of the growth plates15. PP is often present with other morbidities such as McCune-Albright syndrome, congenital adrenal hyperplasia, neurofibromatosis type 1, Sturge–Weber syndrome, adrenal and ovarian tumors12. Premature puberty has been found to be associated with cervical, ovarian and thyroid cancers18. Few studies have reported that early menarche increases the risk of breast cancer in girls20.The incidence of PP is higher in individuals with central nervous system (CNS) disorders or CNS lesions21. Genetic studies suggest that PP has an autosomal dominant mode of inheritance22. Mutations in genes that are involved in sexual development such as MKRN3, KISS1, GPR54, CYP19A1, and LHCGR contribute to PP. Apart from genetic factors, intrauterine growth retardation and low birth weight are linked with early menarche23. It is unclear if childhood obesity is cause or effect of PP23. Exposure to endocrine-disrupting chemicals such as 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), monobutyl phthalate (MBP), n-nonyl phenol (n-NP), t-octylphenol (t-OP), and isoflavones like equol, genistein, and daidzein can influence hormonal dysregulation leading to PP24.The cause of PP can be ascribed to genetic, metabolic, or environmental factors. 70–80% of the variance in pubertal timing can be attributed to genetic factorsSeveral research groups have published valuable information on the causal factors and comorbid conditions associated with PP; this data is worthwhile to collate and analyse systematically to gain further insights on PP.PrecocityDB has an interactive, user-friendly web interface with options to search, browse, and visualize data. The database has curated information of 44 genes and associated 26,874 ontology terms, 235 pathways, and 199 SNPs associated with PP.Home: Short description of PP and information available in PrecocityDB is provided.Quick search: Data available in PrecocityDB for a keyword can be retrieved.Advanced search: Specific queries, through keywords, can be built to retrieve precise information.Search:Browse: Datasets on genes, SNPs, pathways and ontology terms can be screened.SNP visualizer: Allelic variants and SNP coordinates on each chromosome can be visualized. Chromosomes 1 and 6 are associated with the maximum number of SNPs that are associated with PP  followed by LHCGR (luteinizing hormone (LH)/choriogonadotropin receptor gene), KISS1 (Kisspeptin), KISS1R (Kisspeptin receptor), and LIN28B (Lin-28 Homolog B). MKRN3 gene is the most widely studied gene for its association with precocious puberty30. It has ubiqutin protein ligase activity. Mutation in MKRN3 inhibits proteolysis of proteins involved in activation of GnRH neurons31. Mutations in MKRN3, KISS1 and KISS1R contribute to precocious puberty by activating gonadotropic axis32. Inactivating LHCGR mutations in females lead to amenorrhea and infertility, whereas activating LHCGR mutations in males have been associated with familial male-limited precocious puberty (FMPP)33. LIN28B expression influences the timing of major developmental events and is thus associated with pubertal onset. Loss of function mutations in LIN28B gene are known to contribute to precocious development35.Through the curation process, we identified 44 genes and 199 SNPs associated with PP. The highest number of SNPs associated with PP was reported for 40. In order to further validate these observations, disease enrichment analysis was performed based on significant co-occurrences of PP gene-set and diseases using GS2D tool57. It is noteworthy that reproductive, endocrine, metabolic and cancer-related disorders were enriched  and observed that patients with ICCP were prone to develop PCOS in their adulthoodFOXO3 in early pubertal development in rodents45, FOXO gene family is not included in PrecocityDB as its association with PP based on human studies is not yet reported. Interestingly, FoxO signalling pathway was found to be one of the enriched pathways based on enrichment analysis of PrecocityDB genes because of the overlapping genes from related pathways such as insulin signalling pathway, MAPK signalling and PI3K-Akt signalling pathway.Enrichment analysis performed using the genes present in PrecocityDB helped to identify the well-studied pathways of PP Fig. . AdditioPrecocityDB will be updated with time and as more genes get validated for its association with PP; this will lead to more accurate results for enrichment analysis. However, even with the present data the enrichment analysis does give out interesting insights and testable hypothesis for the scientific community.PP adversely affects the quality of life of adolescents and could have long-term health consequences. In order to comprehend the comorbidity risk in PP due to shared genes and biochemical pathways, it is essential to have a high quality database of genes and SNPs that are associated with PP. PrecocityDB is the first online database that catalogues genetic and polymorphism data associated with PP along with relevant reference literature. Pathway enrichment analysis using the PrecocityDB gene-set indicated that individuals with PP are at higher risk of developing reproductive and metabolic disorders such as PCOS, hypogonadism, insulin resistance, metabolic syndrome, glucose intolerance and hyperinsulinism. Pathways related to prostate cancer, breast, endometrial and hormone-dependent neoplasms were also found to be enriched in the PP gene-set. The hypotheses generated based on gene enrichment analysis are consistent with existing clinical observations. The clinical data on longitudinal prognosis for individuals with PP and diseases that are more likely to emerge in adulthood is scarce. This research gap needs to be addressed for evidence-based management of PP.PubMed was searched on 3rd July 2020 using MeSH  terms associated with PP such as, "Precocious Puberties", “Premature Pubarche” , "Puberties, Precocious", "Pubertas Praecox", "Praecox, Pubertas", "Precocious Puberty", "Precocious Puberty, Central", "Central Precocious Puberties", "Central Precocious Puberty", "Precocious Puberties, Central", "Puberties, Central Precocious", "Puberty, Central Precocious", "Sexual Precocity", "Precocities, Sexual", "Precocity, Sexual", "Sexual Precocities", "Idiopathic Sexual Precocity", "Idiopathic Sexual Precocities", "Precocities, Idiopathic Sexual", "Precocity, Idiopathic Sexual", "Sexual Precocities, Idiopathic", "Sexual Precocity, Idiopathic", "Familial Precocious Puberty", "Familial Precocious Puberties", "Precocious Puberties, Familial", "Precocious Puberty, Familial", "Puberties, Familial Precocious", "Puberty, Familial Precocious", "Precocious Puberty, Male-Limited", "Male-Limited Precocious Puberties", "Male-Limited Precocious Puberty", "Precocious Puberties, Male-Limited", "Puberties, Male-Limited Precocious", "Puberty, Male-Limited Precocious", "Precocious Puberty, Male Limited", "Testotoxicosis" and "gene". Using this query, 487 literature records were retrieved from PubMed. These publications were reviewed to confirm gene association with PP  gene association with PP was confirmed based on experiments such as Real Time-Polymerase Chain Reaction(RT-PCR), Restriction fragment length polymorphism (RFLP), Multiplex ligation-dependent probe amplification (MLPA), Single-Strand Conformation Polymorphism(SSCP) and (b) puberty was attained at age less than 8 years for girls and less than 9 years for boys. Studies were excluded if (a) samples were not from human source; and (b) disease terms were not directly associated with PP; such as “pseudo-precocious puberty”, “premature/precocious adrenarche”, “premature/precocious thelarche”, “congenital adrenal hyperplasia”, “McCune Albright Syndrome”, “21-hydroxylase deficiency”, “androgen excess syndrome”, “aromatase excess syndrome”, “hyperandrogenism”, and “hirsutism” Fig. .46, dbSNP47, Ensembl48, UniProt49, GO50, KEGG51, PDB52 and, Reactome53.Relevant data, such as nature of study population, ethnicity, hereditary information, and mutations/SNPs were appended to gene records based on evidence available in literature. The gene records were annotated for additional information such as unique identifiers for gene and protein, protein structures, family and ontology term details, metabolic pathway information by mapping them to databases such as NCBI54, a gene list enrichment analysis tool which is independent of expression data and based on Fisher’s exact test. The transcription factors of the gene-set were identified using TRRUST V255. KEGG database was used as pathway resource. Pathways with adjusted P value < 0.05 were selected. Enriched pathways were mapped to parent pathway groups using KEGG. The pathways were clustered based on KEGG pathway terms using ClueGO v2.5.7 with a minimum of two genes per term56.Pathway enrichment analysis was performed on the manually curated gene-set and its transcription factors using Enrichr57. Diseases with P value and FDR < 0.05 were selected. Enriched diseases were mapped to parent disease terms using a rule-based method adapted from PCOSKBR258.Gene-disease associations were derived for PP gene-set based on statistically significant co-occurrences of genes and diseases in PubMed by using the GS2D toolPrecocityDB was developed using PHP 7.2.24, MariaDB Server 10.1.44, JavaScript, ideogram.js v1.22.0, and XHTML 1.0 and hosted on Apache webserver.59 was used for creation of SNP visualizer. Chromosomes, in haploid state, are shown as vertical bars. Black and grey bands represent heterochromatin region; lighter bands represent euchromatin region; and blue represents variable region. Centromere is marked in pink. Users can select a shape from list of shapes  to display SNPs associated with PP. The gene name, SNP ID and chromosomal location of each SNP can be perused by hovering over the SNP pointer.This tool can be used for visualization of chromosomal coordinates of SNPs associated with PP. Ideogram.js JavaScript librarySupplementary information 1.Supplementary information 2.


The use of IV methylprednisolone has been shown to be associated with some adverse effects. The most feared side effect is acute gastrointestinal perforation and accelerated hypertension particularly during pulse therapy. Hiccups occur less frequently but can cause high levels of discomfort to the patient. In intractable cases, respiratory arrest and death can occur. This article reports the occurrence of hiccups in a patient managed for pseudo Foster-Kennedy syndrome. The hiccups were observed shortly after IV methylprednisolone was administered to the patient and abetted over a period of one week after it was discontinued. Hiccups occur through the neuronal pathway of the hiccup reflex arc, comprising the vagus nerve, phrenic nerve, parts of the sympathetic nervous system (T6-T12), and efferent fibers from the phrenic nerve that supply the glottis and the accessory muscles of respiration. The hiccups resolved with the use of gabapentin. This case report aims to add to the existing body of knowledge of the efficacy of gabapentin in the management of hiccups. Hiccups also known as singlitus occurs due to an involuntary contraction of the diaphragm and the intercostal muscles leading to the inspiration of air, followed by a sudden closure of the vocal cords, producing the “hiccup” sound . They caHiccups are not predominantly found in adults; they can also occur in infants and children. In these cases, hiccups occurrence is usually referred to as self-limited episodes which are due to irritations caused by overeating, eating too fast, immediate change in ingested food temperature, drinking carbonated drinks, etc. These cases abate easily without any clinical significance . HoweverThe neuronal mechanism of hiccups is made up of afferent fibers of the vagus nerve, phrenic nerve, parts of the sympathetic nervous system T6-T12), and efferent fibers from the phrenic nerve that supplies the glottis and the accessory muscles of respiration. The hiccup reflex center is thought to be a spinal reflex originating from C3-C5 segments of the spinal cord or a brain stem reflex originating from an area near the respiratory inhibitory centers in the midbrain -T12, and. StructuThe drugs that are known to cause hiccups in patients include steroids, benzodiazepines, barbiturates, antibiotics, phenothiazines, opioids, and alcohol. Steroids such as prednisolone, methylprednisolone, dexamethasone, oxanodrolone, and progesterone have also been implicated -8.Treatment options for hiccups include the use of pharmacologic and nonpharmacologic agents if its cause can be established. Presently, chlorpromazine is the only drug approved by the Food and Drug Administration (FDA) for the treatment of hiccups because there are no large-scale studies on the efficacy of other regimens for this condition. However, undesirable results have been reported. Other medications such as gabapentin alone (which was used in this report) and baclofen have yielded positive results in the treatment of chronic hiccups , 9.We present a 44-year-old insulin-dependent diabetes mellitus patient who presented with complaints of painless marked deterioration of vision in the right eye of one week prior to presentation. Ocular examination revealed visual acuity of 6/9 in the right eye and hand movement in the left eye. Marked optic disc palor in the right eye and disc edema in the left eye were shown in the optical coherence tomography (OCT). There were also macular exudates, and retinal hemorrhages, see Figure The management of hiccups is challenging due to the lack of sufficient evidence in the use of pharmacologic and nonpharmacologic agents . ChlorprGabapentin has been shown to be effective in managing intractable hiccups . The mecA study by Porzio et al.  reports Also, Campbell et al.  reports Li et al.  reportedAlthough other agents can be used in the treatment of steroid-induced hiccups gabapentin has been proven to yield remarkable results and more reports are needed to support this finding.Steroids are used in the management of several medical conditions and predispose patient to side effects such as acute gastrointestinal perforation and accelerated hypertension, particularly during pulse therapy using high-dose IV methyprednisolone, so clinicians should pay attention to these adverse side effects while treating patients with steroids. In this case report, the adverse effect of IV methyprednisolone administration was hiccups which was abetted with the use of gabapentin; we therefore propose the use of this drug for the control of hiccups.


Age-related macular degeneration (AMD) is the leading cause of severe, permanent vision loss among the elderly in the developed world. The cellular and molecular pathogenesis of initiation and development of AMD remain poorly delineated. The limited resources of the human AMD RPE/choroid tissues impeded the extensive study of the disease. To better understand the molecular and pathway changes in human AMD RPE/choroid tissues, we searched the literature and found three independent studies using high-throughput technology to analyze gene expression in 54 human AMD RPE/choroid tissues and 46 age-matched healthy controls. We downloaded these data, pooled them together, and reanalyzed the difference between molecular and pathways by the Ingenuity Pathway Analysis (IPA) database. Totally, 353 differentially expressed genes (DEGs) were identified, among which 181 genes were downregulated and 172 genes were upregulated in RPE/choroid of AMD patients. Furthermore, several significantly enriched biological processes, including cancer, organismal injury and abnormalities, and ophthalmic disease, were identified associated with these DEGs. By analysis of canonical pathway, the phototransduction pathway and atherosclerosis signaling were the top two significant canonical pathways altered in RPE/choroid tissues in human AMD. As expected, several ophthalmic disease-related molecules, including RHO, PDE6A, 3′,5′-cyclic-GMP phosphodiesterase, and G protein alpha, were in the central nodes of disease network. The bioinformatics technology combined with the existing high-throughput data was applied to evaluate the underlying key genes and pathways in human AMD tissues, which may predict downstream and upstream biological processes and identify potential therapeutic intervention targets in human AMD. Age-related macular degeneration (AMD), also called macular degeneration, AMD or ARMD, is a leading cause of severe vision loss in the elder individuals. The incidence of the disease relates with aging and exponentially increases every ten years after age 50 years . By 2040Retinal pigment epithelium (RPE), a specialized epithelium lying between the choriocapillaris and the neural retina, is initially affected in AMD, resulting in a subsequent loss of photoreceptor cells over time . RPE perElucidation of molecular and pathways in RPE/choroid leading to AMD development and progression in patients is difficult to achieve, as the study involves decade observation. Sometimes the samples can only be acquired after the donor deceased. These studies are also hampered by the need for the patients under surveillance to obtain repeated biopsies. High-throughput gene expression analysis technology, including microarray and RNA-seq, can give us a broad and comprehensive view to the molecular changes in some precious biopsies , such asIn order to generate a comprehensive profile of key genes and pathways altered in human RPE/choroid of AMD, we pooled the three published GEO data . Based on the principle that all the data were from human RPE/choroid tissues of AMD and age-matched healthy controls, three GEO data  were included in the present study. The microarray analysis of GSE50195 was evaluated by Affymetrix GeneChip Human Exon ST 1.0 arrays. The RNA-seq data of GSE99248 were performed on Illumina HiSeq2000 from prepared RNA libraries. The transcriptome profiling of GSE29801 was carried out using the following system 4 × 44 K in situ oligonucleotide array platform Agilent Whole Human Genome.The transcriptome profile datasets involving gene expression in RPE/choroid tissues of human AMD tissues were obtained from NCBI GEO databases (https://www.r-project.org/) and Bioconductor packages (http://www.bioconductor.org/). The quality of samples and data has been well controlled in the original study. For the original array data, the context correction and normalization were performed using the robust multiarray average process. RNA-seq data were downloaded from the GEO database, which have been all normalized, and the reference-based assembly for gene expression calculation has been carried out. Afterward, in the R affy package, they were subsequently converted into expressive measures. DEGs were subsequently identified using the Limma package. The cutoff values for DEG screening using the Benjamini and Hochberg procedure were considered to be p ≤ 0.01 and absolute Log2Ratio ≥1. The tables of the original differentially expressed gene of GSE29801 were downloaded. The cutoff values for DEG screening were set as p ≤ 0.01 and absolute Log2Ratio ≥1. The quality of samples and data has been well controlled in the original study. Finally, using the Bioconductor package Venn diagram, the differentially expressed genes among GSE50195, GSE99248, and GSE29801 in RPE/choroid between AMD patients and controls were identified. The cutoff values were considered to be p ≤ 0.05 and absolute Log2Ratio ≥1.The GSE50195 and GSE99248 were reanalyzed using the R software .Ingenuity pathway analysis (IPA) (Qiagen) has been used to identify top biological functions, as well as canonical pathways associated with DEGs from GSE29801, GSE50195, and GSE99248. Fisher's exact test was carried out to calculate the probability if any biological function or canonical pathway could be explained by chance alone. Algorithmically, molecular interaction networks are created based on the connectivity of the molecules. Network scores were determined using Fisher's exact test and corresponded to −log 10  of the biological processes involved, a z-score was measured. A p < 0.01 and absolute Log2Ratio ≥1 as the cutoff. Duplicates were consolidated using the maximum absolute log ratio. Finally, 353 DEGs were identified, among which 172 were upregulated (p ≤ 0.01 and Log2Ratio ≥1) and 181 (p ≤ 0.01 and Log2Ratio ≤ −1) were downregulated , organismal injury and abnormalities , and ophthalmic disease  were identified as the top three significantly enriched terms in the perspective of diseases and disorders. Cell morphology , cellular movement , and cellular compromise  were found as the top three significantly enriched terms in the perspective of molecular and cellular functions. Nervous system development and function , embryonic development , and organ development  were shown as the top three enriched terms in the perspective of physiological system development and functions.Ingenuity pathway analysis (IPA) (Qiagen) was used to explore the biological functions enriched in the DEGs. Several significantly enriched biological processes associated with DEGs were identified by IPA . Cancer p< 0.05. p value) = 12.536) and atherosclerosis signaling  = 5.024) were the top two major pathways altered in RPE/choroid tissues in human AMD. The complete list of the IPA-identified pathways is given in Supplementary p values and Log2Ratio for each gene in top ten pathways are listed in Supplementary Canonical pathway analysis could inform the key metabolism and signaling pathways in which the DEGs may be involved. A total of 27 significant pathways were identified using the cutoff of RHO gene codes the protein rhodopsin, which is found in specialized light receptor cell rods and necessary for normal vision. Several RHO polymorphisms and haplotypes have been reported and confer remarkable sensitivity to AMD [α, β, and two γ subunits [PDE6A are one of the most common causes of arRP[The molecular interaction networks were further generated using the connectivity of the identifiedDEGs and ranked by identified score. We suggested 23 networks, which were generated by the identified DEGs  and decreased survival of organism  were the most affected subcategories.Biological processes and functions that are likely to be affected by the DEGs were analyzed by downstream effects analysis. Furthermore, downstream effects analysis also predicted the increased or decreased status of the biological processes. In the present study, both the z-score and rocesses . The incp value ≤ 0.05 were used as the cutoff values. Excluding the chemicals and drugs, 32 upstream regulators were found, in which 7 upstream regulators were predicted to be inhibited while 27 upstream regulators were predicted to be activated. The molecular types of these upstream regulators were mainly cytokines, transcription regulators, complex, and enzyme. Notably, the immune and inflammation molecular, such as IFNa/β, IL12, CCL2, TLR7, and IFNA2,were identified in the upstream regulators, which highlight the importance of the immune response in the pathogenesis of AMD  plays a key role in ocular development and maintenance of retinal homeostasis but has insufficient representation in large-scale gene expression and function datasets, especially in AMD human tissues. Although there were several gene expression and function studies in human AMD RPE/choroid tissues, neither of them were based on global gene expression throughput transcriptomic screening , 20, norIDO1 and TDO2, two key genes in tryptophan metabolism, recently were suggested to associate with autoimmune disease and tumor progression [RHO, CADM1, ELOVL4, GNAT1, GUCY2F, RP1, and SLC24A1 were inductive in this group. Most of them play an important role in development and metabolism in retinal rod and cone photoreceptors by mediating the protection of the retina from nutrient delivery, oxidative stress, ionic homeostasis, growth factors release, and ocular immune privilege establishment [Bioinformatic tools, such as IPA, can be used to identify gene modulations . In thisgression , prompt lishment , 25–27. CNGA1, CNGB1, GNAT1, GNAT2, GNGT1, GUCY2F, OPN1LW, PDC, PDE6A, RCVRN, RGS9, RHO, and SAG were found in the DEGs. The second top altered pathway is atherosclerosis signaling. Although AMD risk variants in the genome-wide association analysis are also involved in atherosclerosis [The RPE is essential for the photoreceptor's normal function and survival, although it is not the part of neural retina . The RPEclerosis , 32, it “Hereditary disorder, neurological disease, and organismal injury and abnormalities” changed mostly in human RPE/choroid tissues of AMD, which was found by enriched network analysis. In these networks, RHO, PDE6A, 3′,5′-cyclic-GMP phosphodiesterase, and G protein alpha were in the central “nodes,” which prompt their roles as the target genes for AMD treatment. Interestingly, these molecules are being investigated as potentially potent pharmacological targets in AMD treatment. Rho-associated kinase (Rho-kinase/ROCK) inhibitors for glaucoma have already been clinically applied  and wideβ, IL12, CCL2, TLR7, and IFNA2, which highlight the immune response in the initiation and progression of AMD. Indeed, AMD has been recognized as an immune-related disease like Alzheimer's and cardiovascular disease [AMD lesions have shown through histopathological evaluation the involvement of inflammatory cells, including lymphocytes, macrophages, and mast cells , which i disease . FurtherIn summary, we performed a comprehensive analysis of the existing high-throughput gene expression datasets in RPE/choroid of AMD. Although there were only a few genes overlapped, probably because of the heterogeneity of AMD, we identified shared pathways and networks made up by the DEGs of these datasets. Our results provided a panorama for better understanding the role of RPE/choroid in the pathogeniesis of AMD. Further, our study might create new avenues of AMD therapeutic strategy by manipulation of these key genes and critical pathways in human RPE/choroid tissues.


Plants using the C4 photosynthetic pathway have an efficiency advantage, particularly in hot and dry environments. They account for 23% of global primary production and include some of our most productive cereals. While previous genetic studies comparing phylogenetically related C3 and C4 species have elucidated the genetic diversity underpinning the C4 photosynthetic pathway, no previous studies have described the genetic diversity of the genes involved in this pathway within a C4 crop species. Enhanced understanding of the allelic diversity and selection signatures of genes in this pathway may present opportunities to improve photosynthetic efficiency, and ultimately yield, by exploiting natural variation. Here, we present the first genetic diversity survey of 8 known C4 gene families in an important C4 crop, Sorghum bicolor (L.) Moench, using sequence data of 48 genotypes covering wild and domesticated sorghum accessions. Average nucleotide diversity of C4 gene families varied more than 20-fold from the NADP-malate dehydrogenase (MDH) gene family (θπ = 0.2 × 10−3) to the pyruvate orthophosphate dikinase (PPDK) gene family (θπ = 5.21 × 10−3). Genetic diversity of C4 genes was reduced by 22.43% in cultivated sorghum compared to wild and weedy sorghum, indicating that the group of wild and weedy sorghum may constitute an untapped reservoir for alleles related to the C4 photosynthetic pathway. A SNP-level analysis identified purifying selection signals on C4 PPDK and carbonic anhydrase (CA) genes, and balancing selection signals on C4 PPDK-regulatory protein (RP) and phosphoenolpyruvate carboxylase (PEPC) genes. Allelic distribution of these C4 genes was consistent with selection signals detected. A better understanding of the genetic diversity of C4 pathway in sorghum paves the way for mining the natural allelic variation for the improvement of photosynthesis.C This reaction produces toxic phosphoglycolate which has to be converted to useful metabolites requiring substantial metabolic energy [2 is termed photorespiration. Photorespiration becomes a major constraint of photosynthesis in situations where CO2 to O2 ratios are low and temperatures are high. The evolution of C4 photosynthesis coincided with declining atmospheric CO2 concentrations [2 in the vicinity of ribulose-1,5-bisphosphate carboxylase (Rubisco).Cant taxa . The maic energy . This watrations ,4 as a m4 plants, this is achieved via spatial separation of the initial CO2 fixation and the Calvin–Benson–Bassham cycle in two different cell types, most often mesophyll cells and bundle sheath cells [2 concentration in C4 bundle sheath cells is up to 10-fold higher than that found in C3 mesophyll cells [4 photosynthesis is not only more efficient compared with C3 photosynthesis in terms of reducing energy losses from photorespiration, but due to the improved efficiency of this pathway, it renders plants more nitrogen- and water-use efficient [4 plants are more productive than C3 plants in areas with high light intensities, warm temperatures, and low rainfall, such as the sub-tropical and tropical areas around the globe.In the majority of Cth cells . CO2 conll cells . At highfficient ,8. C4 plmiscanthus, and switchgrass, use the C4 pathway [4 crops account for an estimated 23% of global primary production [4 photosynthesis pathway is a good starting point to improve photosynthetic capacity and resource efficiency in crop plants. Attempts are currently being undertaken to integrate characteristics of the C4 pathway into C3 crops [Many of the major crops that originated from warm and dry regions of the world, such as maize, sorghum, millet, sugarcane,  pathway . C4 cropoduction . Improveoduction . The C4 C3 crops ,12,13,144 photosynthesis in different plant taxa [4 species in terms of the biochemical pathway. It has long been known that three major biochemical subtypes—nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME), nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) and phospho-enol-pyruvate carboxykinase (PCK)—exist among C4 species [4 cereals belong to, differences in pathway and performance are likely to exist, as C4 photosynthesis has evolved at least 25 times in this group of plants [4 photosynthetic efficiency [However, possibly due to multiple independent evolutions of Cant taxa , large v species . More re species  and that species . Especiaf plants . Explorificiency .4 crop well-known for its adaption to drought and high temperatures. It provides staple food for over 500 million people in the semi-arid tropics of Africa and Asia; in addition to being an important source of feed, fiber, and biofuel. Due to these characteristics, it is expected to play an increasingly important role in meeting the challenges of feeding the world’s growing population under the threat of global warming. Substantial variation in photosynthesis and related traits has been revealed in sorghum [Sorghum is an NADP-ME subtype C sorghum ,21,22,234 photosynthetic pathway. Several high-throughput comparative transcriptomics and evolutionary studies using C3 and C4 phylogenetically related species and cell-specific gene expression have elucidated the key genes and regulatory networks that underpin the C4 photosynthetic pathway [4 genes, mined their allelic diversity and investigated signatures of selection during domestication in sorghum.The recent assembly of whole-genome sequences for a wide range of wild and cultivated sorghum species ,25,26 pr pathway ,35,36,374 genes), and their non-C4 isoforms in sorghum were extracted from two previous studies [4 isoforms are homologous of C4 genes but there was no evidences supporting their involvement in the NADP-ME photosynthetic pathway. Homology between these sorghum C4 genes and their non-C4 isoforms was further verified via a local blast strategy. Protein sequences of these 9 core C4 genes were extracted from the sorghum reference genome V3.1 and were blasted against the reference genome. Blast hits of each gene were filtered using the criteria: E-value <−10, sequence identity >60%, and alignment length >80%. All hits of the same gene satisfying the criteria were plotted based on –log ; only hits of top –log  class were considered if clear differentiation among them was visualized, otherwise all hits were used.This study focuses on 8 key proteins in the NADP-ME photosynthetic pathway in sorghum . A total studies ,39  reported in previous studies [Sequence data of the identified C studies ,25,26. TST [Population genetic parameters including nucleotide diversity (θπ) , Tajima’ST , which mST . The BioST between the group of cultivated sorghum and the group of wild and weedy sorghum > 95% of the population pairwise distribution, (3) Tajima’s D < 0. Criteria for balancing selection included: (1) θπ and hW > 25% of the empirical distribution in the cultivated group, (2) FST between the group of cultivated sorghum and the group of wild and weedy sorghum < 90% of the population pairwise distribution, (3) Tajima’s D > 5% of the empirical distribution.The criteria used in Mace et al. (2013) were employed to identify genes under purifying selection and balancing selection, respectively. Criteria for purifying selection included: (1) θπ and hW < 5% of the empirical distribution in the cultivated group, (2) FST between the group of cultivated sorghum and the group of wild and weedy sorghum were computed for these 27 genes using CDS sequence in PopGenome, a population genomics package implemented in the R environment (http://cran.r-project.org/) [ST, and Tajima’s D for each single nucleotide polymorphism (SNP), respectively, with a slide window of 1-bp and 1-bp step size. Functional annotation of each SNP was conducted using get.codons command. Fold decrease of θπ in the cultivated sorghum group compared to the group of wild and weedy sorghum was calculated to represent reduction of diversity (RoD). The following criteria were adopted to identify sites with signature of purifying selection: (1) A RoD greater than the average of neutral genes; (2) FST > 0; (3) Tajima’s D < 0. The following criteria were adopted to identify sites with signature of balancing selection: (1) An increase in diversity (IoD) in the cultivated group and the group of wild and weedy comparison; (2) FST > 0; (3) Tajima’s D > 0.Population genetics parameters including θπ, Tajima’s D, and Fct.org/) . Specifi4 gene families using the neighbor-joining method with default settings  in Geneious 8.1.2 . Analysis of haplotype network was conducted using a combination of the R package ape [Guinea margaritiferum and S. propinquum  . Nucleotide diversity varied dramatically among the C4 gene families, with the NADP-MDH genes displaying the lowest levels of diversity across all genotypes (average θπ = 0.25 × 10−3), followed by NADP-ME genes (θπ = 0.93 × 10−3), PPCK genes (θπ = 1.20 × 10−3), PEPC genes (θπ = 2.11 × 10−3), CA (θπ = 2.26 × 10−3), and PPDK-RP (θπ = 2.96 × 10−3), while PPDK genes showed the highest level of diversity (θπ = 5.21 × 10−3)  A. The on4 genes with non-C4 isoforms in each gene family with the average overall genetic diversity of C4 genes being comparable to that of their non-C4 counterpart (4 PPDK-RP gene (Sobic.007G166300) and C4 NADP-MDH gene (Sobic.002G324400) had an overall θπ which was 161.76% and 79.85% higher than their non-C4 isoforms, respectively, whereas the θπ of the C4 PPDK gene (Sobic.009G132900) was 75.16% lower than that of the non-C4 PPDK isoform. Nucleotide diversity of C4 genes in the other gene families was within the range of variation of their non-C4 isoforms.Mixed trends were found when comparing Cnterpart . The C4 4 gene families was significantly reduced during sorghum domestication . Averaged across all C4 gene families genetic diversity was reduced by 22.44% in the domesticated compared with the wild and weedy group and when just the 9 core C4 genes were considered, the reduction was 22.98%. However, the reduction of genetic diversity during domestication in C4 genes was not significantly different from that in housekeeping genes . Among the 27 genes, Sobic.003G292400, a non-C4 NADP-ME isoform, exhibited the most severe reduction in genetic diversity, with a reduction of 98.23%. The C4 version of that gene, the NADP-ME gene (Sobic.003G036200), showed the greatest loss of genetic diversity (51.89%) among the C4 genes, with an FST between the cultivated and wild and weedy groups of 0.06 (4 isoform of NADP-ME (Sobic.009G069600), a non-C4 isoform of PPCK (Sobic.006G148300), and a non-C4 CA isoform (Sobic.003G234600) showed a more than 2-fold increase in genetic diversity in the cultivated group.Genetic diversity across Cng genes  , only one gene  was identified as being under balancing selection, which maintains diversity of selected genes, during sorghum domestication, while no gene was identified as being under purifying selection, which reduces diversity of selected genes (4 PPDK-RP isoform (Sobic.002G324700) to 0 in the C4 PPDK (Sobic.009G132900). The C4 PEPC gene (Sobic.010G160700) had the highest number of non-synonymous SNPs (9) among the 9 C4 genes  and CA (Sobic.003G234200), three of 4 non-C4 NADP-ME , both two non-C4 PPDK-RP , and a non-C4 PEPC gene (Sobic.007G106500). Among the 2 C4 genes with SNPs under selection, Sobic.009G132900 had 3 synonymous SNPs under purifying selection, while Sobic.003G234200 had a non-synonymous SNP under purifying selection.The selection signature of these Ced genes . SubsequC4 genes . In contC4 genes . Genes w4 PPDK (Sobic.001G326900) had 24 SNPs under balancing selection including 5 non-synonymous SNPs, and additionally had an overall gene-level signature of balancing selection based on the previous analysis. Two C4 isoforms, PPDK-RP (Sobic.002G324400) and PEPC (Sobic.010G160700), were identified with 3 and 2 SNPs under balancing selection, respectively, although none of them were non-synonymous SNPs. Two non-C4 PEPC  were identified with SNPs under balancing selection, with Sobic.003G100600 having 21 SNPs including 2 non-synonymous SNPs exhibiting signatures of balancing selection. The other 2 genes with SNPs under balancing selection were a non-C4 CA isoform, Sobic.002G230100, and a non-C4 PPCK isoform, Sobic.004G219900.A total of 60 SNPs across 8 genes were identified as being under balancing selection, 7 of which were non-synonymous SNPs distributed across 2 genes . The nonS. propinquum, e.g., 4 unique haplotypes were observed for the C4 isoform of PEPC, with the 2 S. propinquum accessions sharing a single private haplotype. To investigate allelic variation of 4 core C4 genes with SNPs under selection in sorghum, haplotype networks were constructed using CDS SNPs. Based on 16 SNPs within the CDS of the PPDK gene (Sobic.009G132900), 8 haplotypes were identified. Five haplotypes were identified in the wild and weedy genotypes, with 3 being private haplotypes and two of them being maintained in cultivated sorghum; two new haplotypes arose in cultivated sorghum after domestication (A phylogenetic tree was constructed using the CDS of these 27 genes to depict the genetic relationship of 48 accessions . The inttication A. Ten hatication B. The lo4 PEPC gene (Sobic.010G160700), 4 haplotypes were identified. Wild and weedy genotypes encompassed 3 haplotypes and all of them were maintained in cultivated sorghum  had 22 SNPs in the CDS, based on which 5 haplotypes were identified. Two haplotypes were characterized by the wild and weedy genotypes, with the main wild haplotype maintained and further diversifying into two new haplotypes in the cultivated group C. Based  sorghum D. S. pro4 photosynthesis has been studied extensively at the cross-species level with signals of adaptive evolution identified on key genes in the C4 pathway [4 photosynthesis is driven by environments characterized by low CO2 availability, such as hot and dry environments in which CO2 uptake is limited by stomatal closure, it is likely that within-species adaptive variation also exists. However, to our knowledge, studies of within-species allele diversity and signatures of selection on key genes in the C4 pathway have not previously been undertaken.The evolution of C pathway ,48,49,504 photosynthesis. In this study, we performed the first investigation of the genetic diversity of C4 gene families within a C4 species using a collection of 48 sorghum lines. We focused on 9 C4 genes due to their reported key roles in C4 photosynthesis. Our collection of sorghum represents all major cultivated sorghum races, landraces, and wild progenitors, and captures a good proportion of genetic diversity within sorghum. Substantial variation of nucleotide diversity was observed among these 8 C4 gene families in sorghum, with the NADP-MDH gene family showing the least diversity and the PPDK gene family showing the greatest diversity. Nine core C4 genes also exhibited varying degrees of genetic diversity, ranging from θπ values of 5.04 × 10−3 and 4.32 × 10−3 in PPDK-RP and rbcS to θπ values of 0.33 × 10−3 and 0.67 × 10−3 in NADP-MDH and NADP-ME. However, despite such low levels of diversity, non-synonymous SNPs were identified in both NADP-MDH and NADP-ME , indicating the function of this gene is highly conserved.Knowledge of existing natural variation and levels of genetic diversity is a pre-requisite for the optimization of C NADP-ME . C4 PPDK4 gene families, indicating that wild sorghum, as a repository for genetic diversity, might harbor alleles useful for improving C4 photosynthesis.Cultivated sorghum was domesticated more than five thousand years ago in Africa ,52,53. T4 gene families was not significantly different from the genome-wide average, indicating that this gene family has not been under particularly strong selection pressure. Similarly, none of the 9 core C4 genes showed a domestication signal at the gene level. The absence of large sequence variation at the gene level is also consistent with previous evolutionary studies suggesting that relatively minor changes to pre-existing regulatory networks and the use of pre-existing cis-elements were often sufficient to recruit genes into the C4 pathway [4 isoform of the NADP-ME gene found in maize and sorghum is one such gene that has been found to be activated for C4 photosynthesis via subtle changes to its promoter, while the rest of the gene is highly conserved [However, the overall reduction in diversity of C pathway ,57. The onserved . This is4 genes, PPDK (Sobic.009G132900) and CA (Sobic.003G234200), with SNPs under purifying selection, while the other 2 C4 genes, PPDK-RP (Sobic.002G324400) and PEPC (Sobic.010G160700), were identified with SNPs under balancing selection. Previous studies have demonstrated that SNP-level analysis using less stringent criteria is superior for capturing soft selection signals compared with genome-wide ranking [A further high-resolution investigation of domestication signature at the SNP level revealed 2 C ranking ,58. Howe ranking  and the 4 isoforms of PPDK and PEPC were also found to show signals of positive selection in a previous cross-species evolutionary study using orthologous groups from closely related C3 and C4 grass species including sorghum [2 assimilation rate [4 pathway and its residues and regions involved in catalyzes are highly conserved in C4 species [4 isoform of the PPDK gene in our study.The C sorghum . PPDK anion rate , especiaion rate ,63. Howe species , possibl2, and variation of its affinity for CO2/HCO3− amongst species has been documented [4 photosynthesis as it catalyzes the first step of the C4 pathway, converting CO2 to HCO3− [4 dicot Flaveria bidentis, where antisense plants with <10% of wild-type CA activity required high CO2 for growth and showed reduced CO2 assimilation rates [PEPC is also regarded as a potential limiting step in the assimilation of COcumented ,66,67. Cto HCO3− . It was on rates ,70. Receon rates .4 pathway was indirectly improved during sorghum domestication. Without photosynthetic rate being a direct selection target in breeding programs, a steady increase in leaf photosynthetic rate over time of cultivar release has been shown in other cereals, e.g., in Australian bread wheat [4 PPDK-RP and PEPC may reflect adaptation to diverse environments, as both PPDK-RP and PEPC are associated with abiotic stress [4 genes all showed selection signals contrasting with their C4 counterparts with both two non-C4 PPDK-RP  containing SNPs under purifying selection and the non-C4 PPDK (Sobic.001G326900) containing SNPs under balancing selection.The signal of soft purifying selection on PPDK and CA may suggest the Cad wheat . The balc stress ,74. Inte4 genes in cultivated sorghum does not mean these haplotypes are inferior in terms of photosynthetic efficiency, as photosynthesis was not specifically targeted during sorghum domestication [After domestication, sorghum was introduced from tropical to temperate areas, and adapted to divergent local environments. New mutations also arose during this diversification process, and played an important role in local adaptation. In the haplotype analysis, these haplotypes unique to cultivated sorghum are likely to be young alleles arising after domestication, while haplotypes unique to the wild progenitor indicate that some haplotypes were lost during domestication of sorghum. Nevertheless, the loss of wild haplotypes of Ctication . On the 4 photosynthesis has been well studied over the past 50 years and key components of this complex pathway have been identified following the advent of transgenic and sequencing technologies [4 pathway is an important step towards mining the natural allelic variation for the improvement of photosynthesis. Cnologies . UnderstFurther investigation of these allelic variation to link them with agronomical traits will provide new targets for sorghum improvement .


The occurrence and importance of the C4 pathway remains uncertain, however. Here, we demonstrate that carbon fixation in Ulva prolifera, a species responsible for massive green tides, involves a combination of C3 and C4 pathways, and a CA-supported HCO3− mechanism. Analysis of CA and key C3 and C4 enzymes, and subsequent analysis of δ13C photosynthetic products showed that the species assimilates CO2 predominately via the C3 pathway, uses HCO3− via the CA mechanism at low CO2 levels, and takes advantage of high irradiance using the C4 pathway. This active and multi-faceted carbon acquisition strategy is advantageous for the formation of massive blooms, as thick floating mats are subject to intense surface irradiance and CO2 limitation.Most marine algae preferentially assimilate CO Ulva prolifera takes place via a combination of C3 and C4 pathways in combination with the enzyme carbonic anhydrase. The active and multi-faceted carbon acquisition strategy in U. prolifera is advantageous for the formation of massive blooms as the thick floating mats are subject to intense surface irradiance and CO2 limitation.Liu et al. present evidence that carbon fixation in  Rubisco , a key C3 enzyme responsible for carbon fixation, requires inorganic carbon in the form of CO22. However, the demand for CO2 in algal photosynthesis is generally higher than CO2 concentrations in natural seawaters, e.g., the half-saturation constants for CO2 of Rubisco in diatoms are 30–60 μM3 but CO2 concentrations in natural seawater are only 5–25 μM4. Thus, CO2 can be an important factor limiting algal proliferation in the ocean.The Calvin–Benson cycle (C2-concentrating mechanisms (CCMs) that enable accumulation of CO2 via bicarbonate (HCO3−) enzymolysis5. A dominant route in the CCMs is that intra- or extracellular dehydration of HCO3− is catalyzed by carbonic anhydrases (CA) to release CO2 to increase reactions at the Rubisco site  and PEPCase kinase (PEPCKase). CO2 incorporated in this manner eventually enters the C3 cycle to increase reactions at the Rubisco site  gene and enzyme analysis in U. prolifera revealed the existence and activity of the C4-related enzyme pyruvate orthophosphate dikinase (PPDKase)15; and (2) tissue δ13C range of U. prolifera (−21.9 to −14.9‰) at 16 stations in the Yellow Sea indicated a mix of C3 and C4 pathways in carbon fixation16.4 pathway in U. prolifera. That pathway requires the participation of the key C4 enzymes, PEPCase and PEPCKase. PEPCase (in the cytoplasm) supports catalysis of carboxylation of phosphoenolpyruvic acid (PEP), which uses HCO3− as the inorganic carbon substrate leading to C4 compound synthesis. PEPCKase (in the chloroplast) is a decarboxylase, generating CO2 and insuring efficient transfer of CO2 to Rubisco17. PPDKase is responsible for catalyzing the regeneration of PEP in the cytoplasm, but it is not the only way for the C4 pathway to acquire PEP17. Some researchers even believe that PPDKase might not assist with net CO2 fixation but rather has a role in protection against photoinhibition18. Therefore, examining the activities of PEPCase and PEPCKase is a necessary step to confirm the existence of C4 pathway in U. prolifera.These two lines of evidence, however, are not sufficient to characterize a full C13C/12C distributions in plant tissue records the integrated pattern of photosynthetic carbon acquisition20; for example, high CO2 uptake leads to low δ13C values , whereas high HCO3− uptake gives high δ13C values . Therefore, tissue δ13C is an important index using to distinguish C3 and C4 activity. The 13C/12C values in seaweeds, however, often display a wide range that makes definition of the C4 contribution uncertain21. The variation in the ratios depends on ambient temperature, light, salinity, nutrients and CO2 concentrations in seawater22. Growth and respiration in seaweeds23 can also lead to discrimination and fractionation of 13C/12C. As canopies of U. prolifera drift in the Yellow Sea, environmental conditions and physiological activity change, forcing variations that may mask clear evidence for C4 activity.The 4 pathway and/or CA mechanism in U. prolifera. Although several environmental factors  may affect the functional expression and activity of CA mechanism and C4 pathway, light intensity and pCO2 are regarded as the most important two under the condition with sufficient nutrient supply24. Massive floating algal mats in nutrient enriched Yellow Sea are not only exposed to strong surface light, but the thick biomass also can reduce the dissolution of CO2 from the air and lead to CO2 limitation. Consequently, the HCO3− systems operated by CA mechanism and/or C4 pathway would likely become active.To resolve this uncertainty, controlled experiments are needed to examine the correlation between the activities of key enzymes and photosynthetic products to ascertain the activity of C3 (Rubisco), CA (extra- and intra-cellular CA) and C4-metabolic enzymes  in U. prolifera. The relationships between the activities of major enzymes and photosynthetic products were analyzed via the corresponding variations in tissue δ13C, aiming to identify the participation of C3 and C4 photosynthetic pathways and CA mechanism in U. prolifera. The results exhibited the coexistence of C3 and C4 pathways and a CA-supported HCO3− mechanism in U. prolifera. The correlated variations of light intensity and pCO2 with enzyme activity indicated that the C4 pathway was most active under high light but that the CA mechanism became active at low CO2 levels. The joint operation of the C4 pathway and a CA-supported HCO3− mechanism in U. prolifera greatly improved efficiency of inorganic carbon fixation and illustrated why massive biomass accumulation can be formed in a short period, when thick floating mats are subject to intense surface irradiance and CO2 limitation in the Yellow Sea.Here, we describe the results of three outdoor culture experiments designed to examine the daily variations of key C3 and C4 enzymes were much higher on a sunny day  under high light intensity , and stayed low activity between 12:00 and 14:00 h, but increased significantly again from 42 to 143 nmol · min−1 · g · fresh weight−1  between 14:00 and 16:00 under reduced light intensity  = 152.08, p < 0.0001, Fig. p = 0.041, 0.001, and 0.011, respectively, Fig. 4 pathway was only detected on sunny days  was induced under high irradiance. Rubisco  did not display a linear relationship with light , C4 enzymes , and CA enzymes (extra- and intra-cellular CA) and their responses to varying light levels were measured over the course of the day .pCO2  and HCO3− concentration , a result that indicated a CA-dominant photosynthetic pathway late in the day when pCO2 decreased. In contrast, extracellular CA was not active and remained unchanged during the experiment  implied an important contribution of HCO3− in carbon fixation in U. prolifera. We cannot specifically define the contribution of HCO3− fixed separately via the CA mechanism versus the C4 pathway, but we found that the environmental factors inducing the activity of C4 enzymes are different from the CA mechanism. The diurnal modalities of C4 enzymes among the three experiments were consistent, although their concentrations were different depending on the ambient environmental conditions at the time of those experiments  at noon, indicating photosynthesis supported by HCO3−  are light-harvesting operators, and the energy flux they capture will be quenched and redistributed between PSII/PSI to achieve a balance and avoid irreversible damage to the photosynthetic systems31. An induction experiment with U. prolifera showed that the electron transport rate (ETR) between PSII/PSI was still high when light intensity reached 800 µmol photons m−2 s−132. Xu and Gao33 found that the ETR and net photosynthetic rate of U. prolifera remain high and stable even as light intensity increased to 2000 µmol photons m−2 s−1. These results explain the continuous carbon accumulation of U. prolifera exposed to light intensity > 1200 µmol photons m−2 s−1 for more than 2 h in sunny day experiments, which is about two- or threefold higher than that required to saturate the photosynthetic rate (600 µmol photons m−2 s−133). PPDKase might play an important role in dissipating excess energy and ATP in cells, since it is responsible for catalyzing the regeneration of PEP in the cytoplasm, where ATP is consumed during PEP formation from pyruvate  when the U. prolifera bloom is forming, dissolved inorganic nitrogen (DIN) concentrations are 10–80 µM near the coast and 1–15 µM offshore16, and the average light intensity in surface seawaters is ~1072.2 µmol photons m−2 s−134. In this study, the DIN concentrations in the culture medium were about 30 µM and the daily average light intensity on sunny days in experiments 1 and 3 was 1196 and 1011 µmol photons m−2 s−1, respectively. These values fit within the range of environmental condition in the Yellow Sea. The pCO2 supply is limited during the formation of green tide in the Yellow Sea, with a range of 300–450 μatm35, and moreover, it can be affected by phytoplankton consumption. Large floating mats formed by U. prolifera block air–sea exchange, thus, HCO3− uptake and assimilation could become important for rapid biomass accumulation. This was reflected in the tissue δ13C 16.Coastal eutrophication and high light supply in the Yellow Sea are important environmental conditions that favor the initiation of CUlva prolifera assimilates CO2 predominately via the C3 pathway, takes up HCO3− via the CA mechanism at low CO2 levels, and takes advantage of high irradiation by deploying the C4 pathway. This adaptive and multifaceted carbon acquisition strategy in U. prolifera is obviously an advantageous biological approach to support fast biomass accumulation and improve oxygen production in the filaments to sustain respiration for algae while floating in thick mats36. The relative contribution of the C4 pathway and CA mechanism in utilizing the HCO3− system and the environmental thresholds that determine which of these pathways dominate at different stages of bloom development, maturity, and decline need to be further elucidated.Ulva prolifera blooms are initiated from the biomass of thalli dislodged in the intertidal zone where the alga is subject to very high diurnal ranges in temperature and light and can tolerate partial desiccation37. Evolutionarily, the expression of C4 photosynthesis in U. prolifera maybe an adaptive response that enables the species to take advantage of rapidly changing conditions, and which also leads to formation of unusual massive blooms. The ability to harness the benefit of C4 photosynthesis might also be a feature of other types of blooming macroalga and warrants investigation.Ulva prolifera were collected from the Yellow Sea and, after cleaning epiphytes off using sterilized seawater, the sampled thalli were cultured in a laboratory incubator for a week prior to the outdoor experiments. Fifty grams of fresh thalli were placed into each of three replicate transparent plastic containers (53.5 cm × 39 cm × 32.5 cm), with 40 L of filtered seawater. To maintain ambient seawater temperatures in the containers, they were suspended in an outdoor seawater pond (60 × 100 m) at Muping Coastal Station of Chinese Academy of Sciences, China. Each experiment lasted 1 day from 08:00 to 18:00, without stirring. The first two outdoor culture experiments were carried out in July 2018, including a sunny day culture (experiment 1) and a cloudy day culture (experiment 2). The two experiments were to examine the responses of major enzyme activities of the C3 and C4 pathways  to diurnal variation of light intensity. The details are given below.Floating specimens of −2 s−1) than cloudy days (10.2–810 μmol photons m−2 s−1).Algal sample was collected every 2 h from each of the three culture containers and stored in a liquid nitrogen tank prior to assays of enzyme activities. Light intensity, salinity, and air temperature were monitored during the culture using a light meter , salinometer , and thermometer , respectively. The initial concentrations of DIN and dissolved inorganic phosphate (DIP) in the culture medium for both the sunny and cloudy day experiments were 34.6 and 0.67 μM, respectively. During experiments 1 and 2, salinity varied from 31.2 to 32.1 and water temperature was not much different on sunny (27.4–32.6 °C) versus cloudy (28.2–31.4 °C) days, but sunlight intensity was much higher on sunny days  was conducted in August 2019 to define the correlation between major enzyme activities and corresponding photosynthetic products. This experiment was carried out on a sunny day, from 08:00 to 18:00. Samples were collected from each of the three culture containers every 2 h and stored in liquid nitrogen for the assays of enzyme activities and tissue δ−1). After the pH in the water sample was stabilized, HCl standard solution was added into the sample to adjust the pH between 3.40 and 3.90. The added HCl volumes and pH values were recorded and total alkalinity calculated as−1); cHCl represents HCl standard solution (mol L−1); VW represents water sample volume (mL); VHCl represents added HCl volume (mL); 38, which was used to calculate the pCO2 and HCO3− concentrations.Twenty-five milliliters of water samples were taken from each of the three culture containers at each 2-h time interval and transferred to a fifty milliliters stoppered polyethylene bottle containing ten milliliters of a hydrochloric acid (HCl) standard solution  were weighed and then soaked in 2 mL of barbiturate buffer . Five milliliters of CO2-saturated water was injected to initiate the reaction. The reaction mixture was cooled in a 4 °C-water bath. The extracellular CA activity was assayed by measuring the time of catalyzed reaction required for the pH to change from 8.3 to 7.3 (Tc). The same procedure was repeated using 2 mL of barbiturate buffer without algal thallus and the uncatalyzed time required for background pH change from 8.3 to 7.3 was recorded (T0). The extracellular CA activity was calculated according to the following formula: Enzyme activity (U/g) = ((T0/Tc − 1) × 10)/algal weight.CA activity was assayed according to the method of Wilbur and AndersonThen, ~0.03–0.04 g of algal thallus was carefully ground in liquid nitrogen and soaked with 2 mL of barbiturate buffer  for the assay of total CA activity. The total CA activity was measured and calculated using the same method and formula as for extracellular CA activity. The intracellular CA activity was calculated by total CA activity minus extracellular CA activity.13C analysis. The samples were analyzed using an isotope-ratio mass spectrometer  at the Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences. Reference gases were calibrated against International Reference Materials . Results are expressed relative to Vienna PeeDee Belemnite. Replicate measurements of a laboratory standard  analyzed with the samples indicated that analytical errors were <0.1‰ for δ13C.After rinsing with 0.1 M HCl and washing with Milli-Q water, algal samples were freeze-dried and ground. Approximately 0.5–1 mg ground sample was placed into a 4 × 6 mm tin capsule for tissue δR2 was used to determine the significance of the results. PEPCKase activity was transformed (χ2) to ensure normality of residuals for the linear regression (Shapiro–Wilk test). Other variables did not require transformation. Two-way analyses of variance  was carried out on data from experiments 1 and 2. These analyses compared the level of enzyme activity under sunny and cloudy conditions and between different time periods (three replicates at each time period). Comparisons for PEPCKase were made across all time periods from 08:00 to 18:00, and for Rubisco and PEPCase for time periods from 08:00 to 16:00. Bartletts test was used to check for homogeneity of variances. A square root transformation was required to correct the variance structure for Rubisco and PEPCase. The 18:00 time period was excluded for Rubisco and PEPCase as variance structure could not be corrected if it was included. Tukey’s HSD was used for the pairwise comparisons. Correlation analyses were conducted on parameters measured in the third experiment for the time period from 08:00 to 14:00  in order to examine the relationships between the activities of key enzymes and photosynthetic products to ascertain the activity of C4 and/or CA pathways in U. prolifera. All analyses were undertaken using Addinsoft’s statistical software XLSTAT.Linear regression analysis was carried out to test the dependence of enzyme activity on irradiance levels using data from both the sunny and cloudy day experiments (experiments 1 and 2). Pearsonʼs Further information on research design is available in the Nature Research Description of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


Together, these findings indicate that icariin exerts its anti-osteoporotic function by inhibiting JNK/c-Jun signaling pathway, and suggest that icariin may be a promising treatment option for osteoporosis.Osteoporosis is a common metabolic bone disease that affects about 40% of postmenopausal women. Treatment options for osteoporosis are limited, however. Icariin is an herbal substance that has been shown to improve bone mass, but the mechanisms are largely unknown. Using bioinformatics analysis, we have identified the hub genes and KEGG pathways shared between icariin-targeted genes and osteoporosis. The top five shared KEGG pathways were the Toll-like receptor signaling pathway, adipocytokine pathway, neurotrophin signaling pathway, NOD-like receptor signaling, and B cell receptor signaling pathway; the hub genes were RELA, NFKBIA, and IKBKB, belonging to the NF-κB family. The identified icariin-targeted genes are involved in inflammation, insulin resistance, apoptosis, and immune responses, and regulate the PI3K-Akt, NF-κB, MAPK, and JNK signaling pathways. Our  Osteoporosis is a metabolic bone disease that leads to an increased risk of bone fractures. Patients with osteoporosis have an impaired bone microarchitecture causing decreased bone mass density and increased bone fragility. Osteoporosis has become one of the major problems of public health . OsteopoIcariin is a natural substance extracted from the herb Epimedium. Icariin has anti-osteoporotic and anti-osteoarthritic effects –7, throuBioinformatics has the potential to identify genes differentially expressed in patients with osteoporosis, predict hub genes based on protein interactions and gene enrichment analyses, and identify the responsible molecular mechanisms , 10. Bioin vitro data show that icariin inhibits apoptosis of human mesenchymal stem cells by suppressing the JNK signaling pathway. Together, our study suggests that icariin may be a promising treatment option for osteoporosis.In this study, we used bioinformatics analysis to identify the icariin-targeted genes and the signaling pathways involved in osteoporosis. Using gene enrichment analysis, shared Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of icariin-targeted genes and osteoporosis were identified, and the hub genes were obtained. Using simulated drawing, our data indicate that icariin exerts its biological effects through regulating the PI3K-Akt, NF-kB, MAPK, and JNK signaling pathways. Our A total of 29 icariin-interacting genes were identified using the Search Tool for Interacting Chemicals (STITCH) database. STITCH is a database that integrates various chemicals into a single resource. Icariin-targeted genes were obtained in STITCH using the default settings. The first shell  was RELA, JUN, PDE5A, NOS3, ESR1, PDE11A, SCARB1, PNPLA2, SLCO2B1, and SLCO1B3. The second shell (protein-protein) was CREBBP, NCOA3, MAPK9, SRC, MAPK8, AKT1, ATF3, MAPK10, FOS, and NFKBIA. The third shell  was IKBKB, NCOA1, BRCA1, NCOA2, SP1, EP300, ATF2, FOSL1, NFKBIB, and sildenafil. The interaction of icariin-targeted genes was constructed using Cytoscape 3.7.2 . Then thp-value ≤ 0.01 were selected for further analyses. Using the miRWalk database, 110 osteoporosis-related KEGG pathways were obtained.Using the Database for Annotation, Visualization, and Integrated Discovery (DAVID), 71 icariin-related KEGG pathways were obtained; 58 KEGG pathways with 26 shared KEGG pathways of icariin-targeted genes and osteoporosis were identified with Venn Diagram . The topThe icariin-targeted genes related to the top 5 shared KEGG pathways are shown in in vitro proliferation of hMSC cells using the cell counting kit 8 (CCK-8). As shown in To investigate the icariin function, we analyzed its effect on To evaluate the molecular mechanisms by which icariin inhibits apoptosis of hMSC cells, we first analyzed phosphorylated levels of c-Jun (p-c-Jun) and c-Jun N-terminal kinase (p-JNK) by western blotting. As shown in Osteoporosis is a metabolic bone disease caused by an imbalance in bone turnover, resulting in an increased risk of bone fractures. The treatment of osteoporosis is still a challenge . IcariinIn this study, we found 26 shared KEGG pathways between icariin-targeted genes and osteoporosis. The top five of the 26 KEGG pathways ranked by increasing p value were the Toll-like receptor signaling pathway, Adipocytokine signaling pathway, Neurotrophin signaling pathway, NOD-like receptor signaling pathway, and B cell receptor signaling pathway. The NF-κB genes RELA, NFKBIA, and IKBKB were identified as the hub genes, because they were involved in all five pathways. The information on chromosomal positions and connectivity of related genes is shown in Using simulated drawing of the icariin-targeted genes related to the five KEGG pathways, our data indicate that icariin exerts its biological effects through regulating the PI3K-Akt, NF-kB, MAPK, and JNK signaling pathways. The identified icariin-targeted genes are associated with inflammation, insulin resistance, apoptosis, antimicrobial peptides, and immune response.The kinases belonging to the JNK family play a critical role in regulating apoptosis . MAPK8, There are many subtypes of osteoporosis, the most common being postmenopausal osteoporosis. In this study, we have only focused on the anti-apoptotic effects of icariin through regulating the JNK signaling pathway. However, since icariin likely regulates other anti-apoptotic pathways, future studies should systematically analyze the mechanisms responsible for its anti-apoptotic effects. In addition, it will be important to analyze its effect in different subtypes of osteoporosis, and compare its efficacy with other anti-osteoporotic drugs.in vitro data indicate that icariin inhibits apoptosis by suppressing activation of JNK and c-JUN. Even though more studies are needed, these data suggest that icariin may become a promising drug for treating osteoporosis.In conclusion, our bioinformatics analysis indicates that icariin exerts its anti-osteoporotic functions through regulating the Toll-like receptor, Adipocytokine, Neurotrophin, NOD-like receptor, and B cell receptor signaling pathways. Our http://stitch.embl.de/). STITCH is a web-based tool used to identify and predict interactions among genes, proteins, and chemicals [The icariin-targeted genes were screened using the STITCH online search tool  bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists , 27. Thep≤0.01). Osteoporosis related KEGG pathways were obtained using Venn Diagram , and the top five shared KEGG pathways with smallest p values were selected.MiRWalk, a comprehensive atlas of predicted and validated miRNA-target interactions , was useThe enrichment information of the top five KEGG pathways was presented with GOplot, an R package for visually combining expression data with functional analysis . Genes ihttps://www.proteinlounge.com).The top 5 shared KEGG pathways with the smallest p values were selected. The KEGG pathways related to icariin-targeted genes were established with the Pathway Builder Tool 20  were obtained from the Orthopedic Laboratory of Tongji Hospital, Tongji University, and cultured in F12 medium  with 10% fetal bovine serum  and 1% penicillin/streptomycin . Lipofectamine® 3000  was used to transfect cells with plasmid RNA oligos. Plasmid-c-Jun and plasmid-JNK  were transfected at a concentration of 50 nM, and 48 hours after transfection, cells were collected for western blotting and qRT-PCR analyses.TRIzol®  was used to isolate total RNA, and the purified RNA was reverse transcribed into cDNA using the ReverTra Ace® qPCR RT Master Mix (Toyobo Life Science). The RT reaction was conducted for 15 minutes at 42°C, followed by 5 minutes at 98°C; the reaction volume was 20 μl. The qPCR thermocycling conditions were: initial denaturation at 95°C for 30 seconds; 40 cycles at 95°C for 5 seconds, and 60°C for 30 seconds; the reaction volume was 25 μl. GAPDH served as an internal control. Relative mRNA expression was normalized to GAPDH, and calculated according to the 2-ΔΔCq method. All experiments were conducted in triplicates.Cell lysates were prepared using NETN buffer  and resolved by SDS-PAGE. Proteins were transferred to PVDF membranes and blocked in 5 % skimmed milk at 4°C overnight. Membranes were probed with primary antibodies and labeled with HRP-conjugated secondary antibodies . Chemiluminescence detection system  was used to visualize the protein bands.Six replicates of 103 hMSCs were added to 96-well plates and cultured for 24, 48, or 72 hours. Subsequently, CCK-8 reagent was added to cells in serum-free medium for 2 hours, followed by measurements of absorbance at 450 nm.Cell apoptosis was determined using Annexin V-FITC/PI apoptosis detection kit  and flow cytometry.GraphPad Prism 8.0  was used to conduct all analyses; the data are presented as the mean ± standard deviation. The Student’s t-test was used to compare two groups of data, whereas groups of 3 or more were compared using one-way analysis of variance with Tukey’s post-hoc test. P<0.05 was considered to be statistically significant. All experiments were performed three times.


To analyze the potential active ingredients and related crucial targets of the ShengDiHuang Decoction (SDHD) formula in the treatment of dysfunctional uterine bleeding (DUB) by using network pharmacology and verification experiment.  In this study, we determined the potential active ingredients from the traditional SDHD formula and their targets with the network pharmacology method. The network of “compound-disease-target” was constructed by the software of Cytoscape. Software of DAVID was used to enrich pathways for these 87 targets of SDHD. Further, the therapeutic effect of SDHD on DUB was verified by observing the morphological changes of the uterus and ovaries and determining the expression of ERS2 and progesterone in the plasma. Rheum and 5 compounds of Rehmannia were selected, and 87 potential targets were screened by network pharmacology. Furthermore, 7 main active ingredients were acquired by the ADME process. In addition, enrichment analysis of drug-target networks indicated that SDHD may play a role in overall coordination through “multicomponent and multitarget” in different organ patterns by regulating multiple pathways directly or indirectly. Finally, in the verification experiment of SDHD on DUB, it was found that SDHD can effectively repair the uterus and ovaries and also have an upregulation effect on the target ESR2 and increase the content of progesterone.  52 compounds of  Overall, this study revealed potential mechanisms of multitarget and multicomponent about SDHD in the treatment of DUB and provided a scientific foundation for further studying the mechanism. For women aged 40 years or more, before menopause, the treatment includes cyclic progestin therapy, oral low-dose contraceptives, levonorgestrel intrauterine device, or cyclic hormone therapy [Dysfunctional uterine bleeding (DUB) is defined as a state of abnormal uterine bleeding , which i therapy . AlthougRehmannia and Rheum (in the weight ratio of 30 : 1), is derived from the recipients of “Qianjinfang” of Sun Simiao in the Tang Dynasty [Rehmannia has the functions of improving hematopoiesis, promoting vascular endothelial cell proliferation, and improving immunity [Rehmannia, as the principle herb, has the effect of cooling blood and hemostasis and tonifying the yin and the kidney, and Rheum as adjuvant herbs assist the effects of Rehmannia to promote blood circulation to remove blood stasis [Traditional Chinese Medicine (TCM) was recognized as a popular complementary and alternative medicine in Western countries , and it  Dynasty . It is m Dynasty . Modern immunity , 12. Rehd stasis . AlthougNetwork pharmacology is a promising strategy for systemic analysis based on interaction networks of diseases-genes-targets-drugs , 15, whiRehmannia was purchased from Baoji Hanfang National Herbal Pieces Co., Ltd. Rheum was purchased from Lanzhou Xukang Pharmaceutical Co., Ltd. Co., Ltd. Gongxuening Capsules were provided by Yunnan Baiyao Group Co., Ltd., hydrocortisone came from Tianjin Jinyao Pharmaceutical Co., Ltd., hydroxycarbamide was from Beijing Jialin Pharmaceutical Co., Ltd., mifepristone was obtained from Zhejiang Xianyi Pharmaceutical Co., Ltd., and misoprostol was from China Resources Zizhu Pharmaceutical Co., Ltd. The ERS2 kit was purchased from Wuhan Huamei Biological Engineering Co., Ltd. The progesterone kit was supplied from the Nanjing Institute of Bioengineering.http://lsp.nwu.edu.cn/tcmsp.php), BATMAN-TCM (http://bionet.ncpsb.org/batman-tcm), and TCM-MESH (http://mesh.tcm.microbioinformatics.org/). Compounds were selected with DL > 0.18 in TCMSP. According to the collected compounds, the potential targets of the ingredients were searched in the database of TCMSP, BATMAN-TCM, and TCM-MESH. Other than these, we also performed the validated targets which were obtained from the HIT database (http://lifecenter.sgst.cn/hit) and the predicted targets which were acquired from the ChemMapper database (http://lilab.ecust.edu.cn/chemmapper/).All compounds of the two Chinese medicinal herbs in SDHD were collected from the TCMSP (http://www.drugbank.ca/) and OMIM (http://www.omim.org/) with dysfunctional uterine bleeding as keywords, as well as obtained from DisGeNET (http://www.disgenet.org/web/DisGeNET/menu/home) using uterine hemorrhage as keywords.Disease-targets were collected from the Drugbank  and then merged and intersected by mapping the Venny (http://bioinfogp.cnb.csic.es/tools/venny/index.html). After that, 52 ingredients of Rheum, 5 ingredients of Rehmannia, and 87 related targets were collected and used as an input to the software Cytoscape (version3.2.1) to construct the predicted-target (PT) network.All the gene names of OB is one of the most important pharmacokinetic parameters in the ADME process . High OBFour topological properties, “degree,” “betweenness centrality,” “closeness centrality,” and “average shortest path length” were analyzed in Cytoscape (version3.2.1) and then the degree values of which are more than two folds of the median degree of all nodes in the network were filtered, which are considered to be the important targets .http://david.abcc.ncifcrf.gov/home.jsp, version 6.8) and p < 0.05 was selected, and then the pathway data obtained from the Kyoto Encyclopedia of Genes and Genomes  were selected.To clarify the pathways which are involved in putative SDHD targets, KEGG and Go enrichment analysis was performed using the Database Visualization and Integrated Discovery software32  rats (230 ± 20 g) were used and supplied by the Chengdu Dashuo Animal Experiment Center. Rats were raised under standard temperature, humidity, and light conditions and fed standard rodent diet and water. The study was approved by the Ethical Committee of the Shaanxi University of Chinese Medicine.Rats were caged with female : male in the ratio of 2 : 1. The next day, the female rats were examined for vaginal suppository and vaginal smear to find whether there is sperm in the vaginal suppository and vaginal smear as the first day of pregnancy. From the first day of pregnancy, all groups were oral administered with hydroxycarbamide at a dose of 450 mg/kg at 8:00 am and injected 3.7 mg/kg of hydrocortisone every day, except for the control group. The treatment was continued for 7 days to make a model of functional dysfunctional uterine bleeding. On the 8th day of pregnancy, mifepristone and misoprostol were administered orally at 8:00 am and at 18:00, respectively. At the same time, a sterile cotton ball was placed in the vagina of each pregnant rat. The next morning, the cotton ball of the rat's vagina was collected, and blood was seen on the cotton ball, indicating that the model was successful. After successful modeling, twenty one successful model rats were randomly divided into the model group, the positive group, and the treatment group with seven rats in each group. The positive group and the treatment group were administered with 0.156 g/kg Gongxuening and 0.0646 g/kg SDHD on the second day for 8 consecutive days, respectively. After the last administration, the rats were fasted with free access to drinking water. On the next day, the rats were anesthetized and blood was taken from the abdominal aorta. Afterwards, the uterus and ovary tissues were taken and fixed in a 10% formalin solution for 24 hours and then sequentially dehydrated with an ethanol gradient, embedded in paraffin, and sliced, and then hematoxylin-eosin (HE) staining was done. The morphological and quantity changes of the uterine glands in the uterus and follicles in ovaries were observed under a microscope. In addition, the determination of ERS2 and progesterone in the serum was performed.Rheum and 8 of Rehmannia. 1085 proteins were predicted for Rheum ingredients, and 319 proteins of Rehmannia ingredients were fished. 415 related targets for dysfunctional uterine bleeding were collected.A total of 65 constituent compounds of the 2 herbs contained in SDHD were collected, including 57 of Rheum, and 5 compounds of Rehmannia were screened. The chemical names, structures, and molecular formulas of all compounds are shown in Supplementary As shown in Rheum meet the requirements of OB ≥ 30%, DL ≥ 0.18, and Caco-2 > −0.40, including aloe emodin (anthraquinone), (−)-catechin (phenols), beta-sitosterol (triterpene), daucosterol (triterpene), eupatin (flavonoid), rhein (anthraquinone), and toralactone (phenols). Although no ingredients in Rehmannia meet the requirements, catapol as the highest content compound in Rehmannia can be considered as the main active ingredient [Through the screening of main active ingredients by the ADME process, we found that seven components in gredient . The valPredicted SDHD target-known therapeutic targets of the dysfunctional uterine bleeding network were constructed using the links between targets of SDHD and known therapeutic targets of disease. As previously reported , if the in vitro [α, plays an important role in female reproductive activity. Related studies have shown that deregulation of estrogen expression may lead to changes in endometrial function [β, is a nuclear hormone receptor and binds estrogens with an affinity similar to that of ESR1 and activates expression of reporter genes containing estrogen response elements (ERE) in an estrogen-dependent manner. It has been well determined that estrogen is involved in the pathophysiology of breast cancer. Estrogen receptor-α can promote the proliferation of cancer tissues, while ER-β can protect against the mitogenic effect of estrogen in breast tissue. The expression of ER-α and ER-β may greatly influence the development, treatment, and prognosis of breast cancer [β have shown that deficiency of ER-β leads to hyperproliferation and loss of differentiation of the uterus [PTGS2 expresses cyclooxygenase (COX-2), and it was reported that cyclooxygenase is the main rate-limiting enzyme in the synthesis of prostaglandins and thromboxane from arachidonic acid, COX-1 is a structural enzyme, and COX-2 is an induced enzyme . Symptomin vitro . Under nin vitro . Currentin vitro . Therefofunction , 29. Thefunction . Abnormafunction . ESR2, et cancer . Previoue uterus , ventrale uterus , and cole uterus . Thence,e uterus , 37. Rele uterus . Angiogee uterus . Factor e uterus . It is re uterus . At the e uterus . So, it p value, and the FDR. As shown in p value less than 0.05 were possible pathways for SDHD treatment of DUB. The results indicated that these proteins are closely related to the immune system, endocrine system, and cancer, such as complement and coagulation cascades, pathways in cancer, estrogen signaling pathway, bladder cancer, proteoglycans in cancer, and hepatitis B. GO analysis involved three main terms, including biological processes (BP), cellular components (CC), and molecular functions (MF). For GO-BP, 89 pathways were obtained (Supplementary p value 0.01, and these pathways are shown in p value less than 0.05 are considered more likely to be involved in the treatment of DUB. As shown in Supplementary p value of less than 0.05, which is shown in In order to understand the mechanism of SDHD on treating DUB and to identify signaling pathways associated with SDHD protein targets, DAVID software was used to enrich pathways for these 87 targets of SDHD. A total of 30 pathways of KEGG were obtained, and the results of all pathways are shown in Supplementary Rehmannia, participates in the activation of thrombus, which significantly prolongs the closure time of whole blood and the occlusion time of platelet embolism, and plays an important role in hemostasis [Rehmannia, has antiplatelet aggregation effect, thereby inhibiting blood coagulation [Rheum, may play a key role in the regulation of pathways in cancer. In the estrogen signaling pathway, estrogen, as a main steroid sex hormone, can regulate a plethora of physiological processes in mammals, including the growth, development, and function of the female reproductive system, bone integrity, cellular homeostasis, and behavior. There is also substantial evidence for an effect of estrogen on the cardiovascular system [α and ER-β in coronary arteries of premenopausal and postmenopausal women. It was found that increased expression of ER-β is associated with advanced atherosclerosis and calcification regardless of age or hormone status [α deletion, the cardioprotective role of estrogen in ischemia/reperfusion injury is lost [α [The pathway of complement and coagulation cascades is a proteolytic cascade in blood plasma and a mediator of innate immunity, a nonspecific defense mechanism against pathogens. There are three pathways of complement activation: the classical pathway, the lectin pathway, and the alternative pathway. All these pathways generate a crucial enzymatic activity, which in turn produces the effector molecules of complement. The main consequences of complement activation are the opsonization of pathogens, the recruitment of inflammatory and immunocompetent cells, and the direct killing of pathogens. Blood coagulation is another series of proenzyme-to-serine protease conversion, culminating the formation of thrombin, the enzyme responsible for the conversion of the soluble fibrinogen to the insoluble fibrin clot. The coagulation system has evolved to limit blood loss at sites of vascular injury, and its biological effects are usually mediated directly by enzymes. In the case of vascular injury, thrombin is produced very rapidly and relatively in large quantities at the site of injury. Therefore, effective inhibitors are needed to ensure that excessive coagulation does not occur . It was mostasis . Acteosigulation . The abogulation . Therefor system . A studye status . Further is lost . Thus, t lost [α .The structure of rat ovary was observed by HE staining. In The morphology changes of uterus are shown in β leads to hyperproliferation and loss of differentiation of the uterus [ESR2, the nuclear hormone receptor, has well determined that estrogen is involved in the pathophysiology of breast cancer. Deficiency of ER-e uterus , ventrale uterus , and cole uterus . It was Progesterone, a female sex hormone produced by the corpus luteum in early pregnancy, induces secretory changes in the uterine lining . It was Rehmannia is less studied, there may be a lack in data collection, and at the same time, the mechanism of action of some targets is still unclear due to less experimental verification. Therefore, we will use more systematic methods in subsequent experiments.At present, we screened SDHD for DUB targets through network pharmacology and combined with experiments, it was found that SDHD further affects the uterine and ovarian damage caused by DUB by improving the reduction of ESR2 and regulates the changes of endometrial tissue by increasing progesterone level. This method and results provided the basis for further mechanism research on SDHD for DUB treatment and also supplied a novel strategy for exploring the molecular mechanisms of traditional medicines. However, since the compound-targets of In this study, the protein targets of DUB and active components of SDHD were collected and then the complex network between drugs and diseases were revealed by using network pharmacology. We found that the pharmacological activities of SDHD on DUB might be associated with the regulation of five main targets that include PTGS2, ESR1, PGR, ESR2, and F10. Also, SDHD has an upregulation effect on target ESR2 and increases the content of progesterone that we have confirmed through experimental verification. However, further experimental validations of these prediction results are required in our future studies.