Ulcerative colitis is a chronic inflammatory bowel disorder that is hard to cure once diagnosed. Bisdemethoxycurcumin has shown positive effects on inflammatory diseases. However, the underlying bioactive interaction between bisdemethoxycurcumin and ulcerative colitis is unclear. The objective of this study was to determine the core target and potential mechanism of action of bisdemethoxycurcumin as a therapy for ulcerative colitis. The public databases were used to identify potential targets for bisdemethoxycurcumin and ulcerative colitis. To investigate the potential mechanisms, the protein-protein interaction network, gene ontology analysis, and Kyoto encyclopedia of genes and genomes analysis have been carried out. Subsequently, experimental verification was conducted to confirm the findings. A total of 132 intersecting genes of bisdemethoxycurcumin, as well as ulcerative coli-tis-related targets, were obtained. SRC, EGFR, AKT1, and PIK3R1 were the targets of highest potential, and the PI3K/Akt and MAPK pathways may be essential for the treatment of ulcerative colitis by bisdemethoxycurcumin. Molecular docking demonstrated that bisdemethoxycurcumin combined well with SRC, EGFR, PIK3R1, and AKT1. Moreover, the in vitro experiments suggested that bisdemethoxycurcumin might reduce LPS-induced pro-inflammatory cytokines levels in RAW264.7 cells by suppressing PI3K/Akt and MAPK pathways. Our study provided a comprehensive overview of the potential targets and molecular mechanism of bisdemethoxycurcumin against ulcerative colitis. Furthermore, it also provided a theoretical basis for the clinical treatment of ulcerative colitis, as well as compelling evidence for further study on the mechanism of bisdemethoxycurcumin in the treatment of ulcerative colitis. Ulcerative colitis (UC) is one of the two major types of inflammatory bowel disease (IBD) that causes recurring and remitting inflammation of the digestive tract . During Curcuma longa. The main components of curcuminoids found in Curcuma longa are curcumin, demethoxycurcumin, and bisdemethoxycurcumin (BDMC) [Curcuminoids, polyphenol coloring compounds, were extracted from n (BDMC) . Comparen (BDMC) ,7. Accorn (BDMC) ,8,9,10. Network pharmacology is a brand-new field that combines high throughput histology, bioinformatics, and system biology, which has been extensively applied to investigate the mechanism underlying the curative action of traditional Chinese medicine . In thisThe MOL2 structure of BDMC was downloaded from the TCMSP (Molecule ID: MOL000940). A total of 240 targets of BDMC were obtained from the PharmMapper server . Furthern = 18), EGFR (n = 16), AKT1 (n = 14), PIK3R1 (n = 14), AR (n = 12), HSP90AA1 (n = 12), CDC42 (n = 10), HRAS (n = 10), ESR1 (n = 10), and RHOA (n = 8), which may be extremely important in the anti-UC of BDMC, were the highest degree-valued target nodes.The string database created a PPI network with 169 edges and 132 nodes, where edges represent protein-protein and nodes represent proteins A. The PPGO and KEGG pathway analyses were performed to investigate the biological functions and pathways linked to the possible anti-UC genes of BDMC. The top 10 significant enrichment terms of biological processes (BP), cellular components (CC), and molecular function (MF) were chosen for presentation A. As sugA BDMC-target-pathway network was established to demonstrate the relationship between BDMC, common targets, and their correlated pathways. As shown in Molecular docking was used to investigate the interaction between BDMC and the four targets . The binding energy is less than 5.0 kcal/mol, indicating that the confirmations have good interactions . As showThe CCK8 assay was utilized to demonstrate the impact of BDMC on the viability of RAW264.7 cells. As shown in To confirm the anti-inflammatory effects of BDMC, the expression of IL-6, IL-1β, TNF-a, and MCP-1 in LPS-induced RAW 264.7 cells were detected by real-time PCR. As presented in The previous results have revealed that lipid and atherosclerosis, the MAPK pathway, and the PI3K-Akt pathway were most greatly linked to the potential anti-UC genes of BDMC. Thus, the phosphorylation of key protein kinases of the MAPK and PI3K-Akt pathways were detected to explore the underlying mechanism of BDMC on the treatment of UC. As presented in A–E, compIn this study, we predicted the mechanism of BDMC in the treatment of UC, based on network pharmacology, and verified those results with in vitro experiments. The results of network pharmacology demonstrated that the underlying mechanism for the BDMC in the treatment of UC is complex, involving multiple protein targets and pathways; SRC, EGFR, AKT1, and PIK3R1 were the top four core potential targets. The molecular docking results showed that BDMC and AKT1 exhibited the best binding interaction. According to the GO and KEGG analyses, the most significant pathways which were involved in the treatment of UC by BDMC were lipid and atherosclerosis, the MAPK pathway, and the PI3K-Akt pathway. Our study is the first study to elucidate the underlying mechanism of BDMC in the treatment of UC using network pharmacology, which provides a theoretical starting point for the clinical treatment of UC.Our study results revealed that multiple genetic and molecular interactions between BDMC and UC via PPI network analysis and drug -target-disease pathway analysis. After a PPI topological screening, SRC, EGFR, AKT1, and PIK3R1 were the top four core targets. It implies that BDMC might be crucial in treating UC by affecting these targets. Furthermore, we performed molecular docking to further verify the candidate core targets. Based on binding potency and hydrogen bonding results , the bonIn addition to the PI3K/Akt pathway, the KEGG analysis also found that target genes were similarly enriched in the MAPK pathway. Several studies have shown that the MAPK signaling pathway is closely correlated with UC. Zhao X et al., demonstrated that the level of P-P38 MAPK was enhanced in UC patients versus control samples . Fan H eAt the same time, our experiment in vitro showed that the protein expression of p-p38 MAPK, p-ERK1/2, and p-JNK were detected, and the expression of p-p38 MAPK was obviously reduced in LPS-induced RAW264.7 cells, while that has no effects on p- p-ERK1/2 and p-JNK. Therefore, we speculated that BDMC may alleviate UC via inhibiting the p-p38 MAPK signaling pathway activation, instead of inhibiting p-ERK1/2 and p-JNK signaling pathway activation. Taken together, our research shows that the PI3K/Akt pathway and p-p38 MAPK pathway may be crucial in the treatment of UC with BDMC.However, there are several limitations to our study. First of all, due to incomplete database information, some critical target genes may be missing. Secondly, various signaling pathways in which BDMC is operating on UC were speculated employing the network pharmacological strategies; howeverthe contribution of each pathway was not identified. Finally, our current findings only provide the direction of future research, whereas the specific mechanisms still need to be carefully investigated in subsequent studies.http://www.lilab-ecust.cn/pharmmapper/, accessed on 1 May 2022) to obtain the targets of the pharmacophore model and 299 targets of BDMC were screened by Norm Fit ≥0.3.The MOL2 structure of BDMC was provided from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), which was inputted into the Pharm Mapper server , Drug Bank , Pharm GKB , Therapeutic Target Database , and Online Mendelian Inheritance in Man . All targets were filtered as “homo sapiens”.Therapeutic targets of ulcerative colitis were offered by five public databases with the keywords “ulcerative colitis”: Gene Cards , with the organism parameter set to homo sapiens, the required minimal interaction ratio of 0.95, and hiding individual target protein nodes, as well as other basic settings left at the default value. The data obtained from the String database were inputted into Cyto-scape3.8.0 to visualize the PPI network. Six topological node metrics were calculated using the CytoNCA plugin in Cytoscape3.8.0, which include network centrality (NC), betweenness centrality (BC), closeness centrality (CC), eigenvector centrality (EC), degree centrality (DC), and local average connectivity (LAC). These parameters may all be used to analyze the characteristics of nodes in an interactive network in-depth. Metrics with higher quantitative values indicate that nodes in a network are more important. To create a brand-new PPI network for further study, target nodes were kept only if all six metrics exceeded their respective medians. The network was handled as a non-directed graph and node degree distribution diagrams were created using the Network Analyzer Cytoscape plugin. Six parameters were also determined for the new network and only nodes having all six parameters greater than their respective median values were kept in place for establishing.The BDMC and UC molecular targets were obtained to construct a PPI network by using the STRING database  analysis and the Kyoto encyclopedia of genes and genomes (KEGG) analysis were employed. Only routes and functional words with q values of less than 0.05 were statistically meaningful and maintained.The software Cytoscape3.8.0 was employed to create a BDMC-target-pathway network and analyze the relationship between compounds, targets, as well as ulcerative colitis.https://pubchem.ncbi.nlm.nih.gov/, accessed on 3 May 2022), then it was imported into Chem Draw 3D to minimize energy through the MM 2 module, and choose the best conformation with the lowest energy, and save it as a mol 2 file. The Protein Data Bank (PDB)  was used to obtain the crystal structures of core protein targets. The BDMC and core protein targets were converted to the PDBQT format using the AutoDockTools software. The AutoDockVina was applied to undertake molecular docking and determine the binding energy. The docking outcome of BDMC and the core protein targets were visualized using the PyMOL software .The 2D structure of BDMC was downloaded from PubChem  was used to evaluate the effects of BDMC  in RAW264.7 cell lines. RAW264.7 cells were seeded at a density of 5 × 104 cells per in-96-wall panel. After, 12 h, the medium was changed to brand-new 1640  that contained various concentrations of BDMC . After 48 h, the culture was stopped, and 10 μL of CCK8 reagent was added, which was then incubated for 1 h at 37 °C. The absorbance at 450 nm was measured using a microplate reader.6 cells per well in 6-well panels. After being incubated for 12 h, the cells were either pre-treated for 1 h with BDMC and an LPS treatment with 1 μg/mL or left untreated for 24 h. Total RNA was extracted from cells using the Total RNA Isolation Kit . Reverse transcription was carried out using the Prime Script TM RT-PCR kit from Takara, Otsu, Japan, and the Biorad CFX Connect  was used for qRT-PCR, as directed by the manufacturer. This research used the following primers: interleukin-6 (IL-6): 5′-CTGCAAGAGACTTCCATCCAG-3′ (forward) and 5′-AGTGGTATAGA-CAGGTCTGTTGG-3′ (reverse); monocyte chemoattractant protein-1 (MCP-1): 5′-CTGTGCTGACCCCAAGAAGG-3′ (forward) and 5′-AGGTGGTTGTGGAAAAGGTAGTG-3′ (reverse); interleukin-1β(IL-1β): 5′-ATGCCACCTTTTGACAGTGATG-3′ (forward) and 5′-TGATGTGCTGCTGCGAGATT-3′ (reverse); tumor necrosis factor alpha (TNF-a): 5′-CGACGTGGAACTGGCAAGAA-3′ (forward) and 5′-GGACCGATCACCCCGAAG-3′ (reverse); and GAPDH: 5′-AGGTCGGTGTGAACGGATTTG-3′ and 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (reverse). The 2−∆∆Ct method was used to calculated relative expressions of mRNAs.RAW264.7 cells were planted at a density of 5 × 106 cells per well, pre-treated for 1 h with BDMC and LPS treatment with 1 μg/mL, or left untreated for 24 h. The Western blot was performed exactly as previously described [p value of less than 0.05 was considered statistically significant.RAW264.7 cells were planted in 6-well plates at a density of 5 × 10escribed . PrimaryWe looked into the potential mechanisms of BDMC using network pharmacology, molecular docking, and in vitro verification. We believe that this research has huge value in terms of providing a theoretical basis for the clinical treatment of UC, as well as providing compelling evidence for further research into the mechanism of BDMC in the treatment of UC.


Fibraurea recisa Pierre (FRP), the classic type of Heat-clearing and detoxifying Chinese medicine, was selected as the object of research.Heat-clearing and detoxifying Chinese medicines have been documented to have anti-Alzheimer’s disease (AD) activities according to the accumulated clinical experience and pharmacological research results in recent decades. In this study, 12 components with anti-AD activities were identified in FRP by a variety of methods, including silica gel column chromatography, multiple databases, and literature searches. Then, network pharmacology and molecular docking were adopted to systematically study the potential anti-AD mechanism of these compounds. Consequently, it was found that these 12 compounds could act on 235 anti-AD targets, of which AKT and other targets were the core targets. Meanwhile, among these 235 targets, 71 targets were identified to be significantly correlated with the pathology of amyloid beta (Aβ) and Tau.In view of the analysis results of the network of active ingredients and targets, it was observed that palmatine, berberine, and other alkaloids in FRP were the key active ingredients for the treatment of AD. Further, Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis revealed that the neuroactive ligand-receptor interaction pathway and PI3K-Akt signaling pathway were the most significant signaling pathways for FRP to play an anti-AD role. Findings in our study suggest that multiple primary active ingredients in FRP can play a multitarget anti-AD effect by regulating key physiological processes such as neurotransmitter transmission and anti-inflammation. Besides, key ingredients such as palmatine and berberine in FRP are expected to be excellent leading compounds of multitarget anti-AD drugs. Alzheimer’s disease (AD) is a degenerative neurodegenerative disease that is progressive, complex, multifactorial, and incurable . AccordiThere has been much evidence indicating that multitarget drug therapy is more effective than one class of drugs for treating complex diseases such as malignancies . DevelopTo solve these problems, we focused on traditional Chinese medicine (TCM). Classical Chinese medicine has been proven to be safe and effective over many years of clinical use . AccordiFibraurea Recisa Pierre (FRP) is a well-known HDTCM that is widely distributed in China, Vietnam, Laos, and Myanmar  was purchased from Su Li Herbal Materials Company , which was authenticated by the Department of Pharmacy of Jinggangshan University by Professor Zhaochang Liang. Several major ingredients of FRP were separated and identified by using methods reported in the literature , the main ingredients and potential action targets of FRP could be obtained. Specifically, a few ingredients with low OB or DL were chosen as candidate ingredients because of their high bioactivity according to the literature. Meanwhile, the UniProt database6 was used to convert the gene name corresponding to each target name and organize the records.terature . Other iPubChem : A databADMETlab 2.0 : A websi7 to predict the relevant physicochemical properties.The common names of the main ingredients of FRP were retrieved from PubChem (see text footnote 1), and all their 2D structures were drawn by ChemDraw 15.0. Then, the SMILES strings of each main ingredients were obtained from PubChem (see text footnote 1) and imported into the ADMETlab 2.0SwissTargetPrediction : A databSearch Server (SEA) : A datab8 and SEA Search Server9 were employed to identify the ingredient-related target proteins according to the determined main active ingredients.Swiss Target PredictionOnline Mendelian Inheritance Database of Humans (OMIM) : A comprDrugbank : A databTTD : A databPharmGKB (Pharmacogenetics and Pharmacogenomics Knowledge Base) : A comprGenecards database : A comprMicrobiology letter platform: An online platform for scientific data analysis and visualization.STRING : A databCytoscape : A powerCytoNCA : A plug-Molecular Complex Detection (MOCDE): A plug-in Cytoscape, which is used to identify sub-modules in the PPI network with biological significance.10 TTD,11 Drugbank,12 PharmGKB,13 and Genecards14 databases, the relevant targets can be obtained. Then, the final targets for the treatment of AD were collected after further processing and removing all duplicates targets. The targets corresponding to the main ingredients of Chinese herbal medicine, FRP, and the treatment of AD were used to obtain the intersection targets using the Microbiology letter platform.15 The intersection data were entered into the STRING database,16 the data were exported in TSV format, and the PPI relationship network was constructed using Cytoscape, thus revealing the visualized network structure map. Then, the core targets were screened using its plug-in CytoNCA, and the common anti-AD target gene clusters and pathway-related target genes of FRP were obtained using MOCDE.By searching “Alzheimer’s disease” and “anti-Alzheimer’s disease” from OMIM,The collated data of the main ingredients and their potential targets of FRP were imported into Cytoscape (1 June 2022) to construct the drug-ingredient-target visualization network diagram. In the process of constructing the drug-ingredient-target visualization network diagram of FRP for AD, each target is constituted as a corresponding node, and in the layout options, a tool-analyzed Network is selected to obtain the degree of each data point. The larger the value of degree, the higher the participation of the node, and the stronger the credibility.DAVID database : A databGene Ontology (GO) is a classification system used to describe gene characteristics, gene product characteristics, and their main functions to describe three specific genes.Biological process (BP) is used to describe the BP in which genes are involved, such as the regulation of growth factors.Cellular component (CC) is used to describe the location of the gene product in the cell, e.g., in the mitochondria and nucleus.Molecular function (MF) is used to describe the function of a single gene product and the function of multiple genes acting together.Kyoto Encyclopedia of Genes and Genomes (KEGG) : A datab17 and GO and KEGG enrichment analyses , their human gene symbols were entered and analyzed by using the AlzData database.AutoDock : A molecRCSB PDB : A power19 and then converted them to 3D and performed energy minimization. Next, the PDB formats of the core proteins corresponding to the core targets were downloaded from the RCSB PDB20 database. Next, water molecules and small molecule ligands were removed from the protein, hydrogen was added, and the active pockets in the 3D structure of the protein were identified by AutoDock Tools. Finally, the prepared protein models and ligand structures were used for molecular docking in the AutoDock Vina software . After performing the molecular docking simulation, the ligands were ranked by their docking scores, the higher the absolute value of the docking score indicates the stronger ability of the main ingredients to bind to the predicted important target proteins, and vice versa.To reveal the binding modes and the strength between active ingredients and their protein targets, molecular docking simulation was performed using AutoDock 4.2. We collected the 2D structures of the main ingredients of FRP by PubChem,via1H NMR , including the topological acute surface area (TPSA) and solubility (LogS) indexes. These results indicated that the main ingredients of FRP possessed good permeability across the cell membrane, as shown in The ADME-related properties of the main ingredients of FRP were evaluated in depth using the online tool ADMETlab 2.0, and all of them conformed to Lipinski’s five laws  showed that these clusters may be relevant to AD treatment. As shown in To explore the mechanism of action of FRP for the treatment of AD, 235 drug-disease intersection gene targets and 12 main ingredients in FRP were used to construct the major ingredient target AD target network. As shown in Gene Ontology enrichment analysis and KEGG pathway enrichment analysis were performed using David on the intersecting targets, and 235 MFs entries were screened out, the 20 closely related MFs are shown in Gene Ontology enrichment analysis got 1,264 entries, of which 947 were BP entries, 111 were CC entries, and 206 were MF entries. As shown in P-value = 1.78E-22) and the PI3K-Akt signaling pathway , which are related to human diseases and are mainly associated with To further evaluate the functions of these overlapping target genes, 235 drug-disease intersection gene targets were filtered through the KEGG pathway. Pathway enrichment analysis revealed that these genes were enriched in 170 signaling pathways, and the top 20 signaling pathways most closely related to AD were the following : AD-relaThe network of main components and their related targets of Chinese herbal medicine FRP is shown in To further validate the relationship between the potential targets of the Chinese herbal medicine FRP and the potential mechanisms of AD (Aβ and Tau), the intersecting genes were entered into the AlzDate database. Among these targets, 71 were associated with Aβ, Tau, Aβ, and Tau , specifiTen targets  were chosen according to the results of the PPI network as the core targets of FRP for the treatment of AD. Therefore, molecular docking was performed by AutoDock software to simulate the interaction between the main ingredients of FRP with these 10 core anti-AD target proteins. The coordinates and box size for molecular docking were depicted in According to the theory of Chinese medicine, AD is considered a multitargeted systemic disease, which is consistent with the contemporary view of modern Western medicine coincidentally . SeveralAlthough network pharmacology has become a useful tool to lend insight into the mechanisms of TCM. Even so, there are still some problems to overcome. Based on the newly bibliometric analysis results of network pharmacology in TCM, determining the key ingredients in TCM  just basin vitro  showed the highest number of anti-AD targets, but the current study on its anti-AD properties is limited. Per the existing literature, palmatine could attenuate LPS-induced inflammation by inhibiting ERK1/2, P38, and Akt/NF-kB signaling pathways . Besidesin vitro . These fin vitro enzymatic activity assay  balance and significantly affect neurotransmitter dysfunction, synaptic dysfunction, and cognitive dysfunction  also reportedly contain some of the same alkaloids compared with FRP (In the further studies, we noted that many other herbal medicines (with FRP . Accordivia multiple pathways, which were concentrated on the neuroactive ligand-receptor interaction, PI3K-Akt, calcium signaling, and cAMP signaling pathways. Meanwhile, 16 core targets were identified by PPI network analysis. According to the extraction experiment and molecular docking results, alkaloids of FRP, such as palmatine and berberine, were not only the most abundant components in FRP but also had the best anti-AD activity. Moreover, these components showed promising enzyme activity against AChE, which currently remains the foremost therapeutic target for AD. In conclusion, the results of this study not only further verified that FRP in AD treatment has the characteristics of “multiple components, multiple targets, and multiple pathways” through network pharmacology but also identified possible leading compounds with potential multi anti-AD activity. Moreover, our research opens the door for the potential application of HDTCM to multitargeted AD therapy. Additionally, future in vivo and in vitro experiments are needed to be performed to verify the results of the present study.In this study, we successfully investigated the key active components and molecular mechanisms of FRP implicated in the treatment of AD by network pharmacology analyses and molecular docking. This research identified 12 ingredients of FRP treatment of AD by targeting 235 targets The original contributions presented in this study are included in the article/YW conceived and designed the review. SsW and YxM wrote the manuscript. YsH, YhH, and YpH revised the manuscript. SsW, YxM, YpH, YsH, YhH, and YW approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.


Isoprenoids are the output of the polymerization of five-carbon, branched isoprenic chains derived from isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP). Isoprene units are consecutively condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate , necessary for the biosynthesis of several metabolites. Polyprenyl transferases and synthases use polyprenyl pyrophosphates as their natural substrates; however, it is known that free polyprenols, such as farnesol (FOH), and geranylgeraniol (GGOH) can be incorporated into prenylated proteins, ubiquinone, cholesterol, and dolichols. Furthermore, FOH and GGOH have been shown to block the effects of isoprenoid biosynthesis inhibitors such as fosmidomycin, bisphosphonates, or statins in several organisms. This phenomenon is the consequence of a short pathway, which was observed for the first time more than 25 years ago: the polyprenol salvage pathway, which works via the phosphorylation of FOH and GGOH. Biochemical studies in bacteria, animals, and plants suggest that this pathway can be carried out by two enzymes: a polyprenol kinase and a polyprenyl-phosphate kinase. However, to date, only a few genes have been unequivocally identified to encode these enzymes in photosynthetic organisms. Nevertheless, pieces of evidence for the importance of this pathway abound in studies related to infectious diseases, cancer, dyslipidemias, and nutrition, and to the mitigation of the secondary effects of several drugs. Furthermore, nowadays it is known that both FOH and GGOH can be incorporated via dietary sources that produce various biological effects. This review presents, in a simplified but comprehensive manner, the most important data on the FOH and GGOH salvage pathway, stressing its biomedical importance The main objective of this review is to bring to light the need to discover and characterize the kinases associated with the isoprenoid salvage pathway in animals and pathogens. Isoprenoids are the most widespread and diverse group of compounds in nature. They are produced by all organisms and are also thought to be some of the most ancient lipids, originating as constituents of the primitive membranes in the first living organisms . All isoThe MVA pathway was the first isoprenoid biosynthesis pathway discovered. It is active in plant cytosol, fungi, archaebacteria, eubacteria, and some protozoa groups ,6. This Regardless of the biosynthetic pathway, IPP and DMAPP can be interconverted by the IPP isomerases. In plants, IPP can be directly used for the production of phytohormones . HoweverIn any case, all polyprenyl pyrophosphates cited previously are employed as substrates for diverse anabolic pathways. For example, FPP and GGPP can be employed for an important post-translational modification of proteins. Typically, protein prenylation occurs when farnesyl or geranylgeranyl moieties are attached to soluble proteins, resulting in their anchoring to membranes. The most studied prenylated proteins are Ras, Rho, and Rap, which are small GTPases involved in cellular signaling and intracellular trafficking ,17. ThisIsoprenoid biosynthesis is the target of some of the most prescribed drugs worldwide, as already cited. For example, statins are employed to treat hypercholesterolemia, as they inhibit mammalian biosynthesis of cholesterol, while bisphosphonates are used in the treatment of osteoporosis due to their ability to accumulate in bone mineral, inhibiting osteoclast activity ,28,29. FAcacia caven, contain great amounts of farnesol (FOH)  CDP and FPP or GGPP, but not with FP. Similar to this, a phytol salvage pathway was described in Arabidopsis thaliana [Plasmodium falciparum); in this system, most probably, those reactions could serve as a mechanism to recycle polyprenols for the biosynthesis of parasitic cofactors. Furthermore, we observed parasites that could use these phosphorylated isoprenoid alcohols, including in Ras and Rap protein prenylation, in addition to condensing them into longer polyprenols and dolichols [In plants, phosphorylation of FOH and GGOH to their respective mono- and diphosphates was first observed in cultures . In thisthaliana . The autolichols .Solanum lycopersicum plants, the FOLK and VTE5 genes encode FOH kinase and phytol kinase, respectively [Synechocystis sp. gene ScVTE5 encodes a phytol kinase [A. thaliana, the AtFOLK gene encodes a FOH kinase [VTE5 gene, by encoding a plastidic phytol kinase [VTE6, by encoding a plastidic phytyl-P kinase [FOLK gene expression was shown to be negatively regulated by the phytohormone abscisic acid. As a consequence, loss-of-function-FOLK mutants displayed an abscisic acid-hypersensitive phenotype and anomalous flower growth under abiotic stress, with the development of supernumerary carpels [VTE5 and VTE6 genes in plants produced accumulations of free phytol and deficiencies in phytyl-PP-dependent pathways, such as tocopherol and vitamin K1 biosynthesis, as well as photosystem I instability and the reduced tolerance to abiotic stress [The consensus, thus far, is that the polyprenol salvage pathway is composed by two independent enzymes: a polyprenol kinase and a polyprenyl phosphate kinase . Howeverectively . The Synl kinase . FinallyH kinase , namely,l kinase ,89, and P kinase . In all P kinase ,88,89,90c stress ,89,90. AVTE5, was identified in mutagenesis studies of plants defective for vitamin E and K1 biosynthesis. FOLK was identified to encode a FOH kinase by similarity with VTE5 [VTE5 and FOLK gene products seem to be related enzymes, despite their different functions. Moreover, both show a modest similarity to dolichol kinase [VTE6 as a phytyl-P kinase was first suggested when an analysis of similarities with bacterial genomes was carried out [In terms of structure, these genes and their encoded proteins show similarities and differences which must be commented on. The first of the genes reported, ith VTE5 . On the l kinase . Remarkaried out . This geS. cerevisiae and E. coli, organisms where polyprenol and polyprenyl-P kinase activities are not naturally found [E. coli allowed for phytol-feeding experiments, followed by the analysis of the product by mass spectrometry [The biochemical characterization of these proteins was not straightforward. The identified polyprenol kinases needed to be recombinantly expressed in ly found ,88,90. Atrometry ,90. AdvaAs mentioned above, the polyprenol salvage pathway was first observed more than 25 years ago, but the enzymes responsible in animals or any pathogen are still unidentified ,80. DespKRAS) are very common in cancer, where they are present in approximately 22% of cases of these pathologies [https://www.cancer.gov/news-events/cancer-currents-blog/2015/turning-off-broken-switch, last accessed on 7 July 2022). Among the strategies devised, inhibitors of the MVA pathway for the treatment and prevention of different types of cancers have been explored for decades. However, the use of statins mostly shows an unsatisfying ability to treat and prevent cancer [Oncology is probably the main area where polyprenols have been found to exert a dramatic role. This is somewhat unsurprising since isoprenoids are required for several cancer-related processes such as the Hedgehog pathway, the nuclear localization of the Yes-associated protein, steroid hormone formation, and transcriptional co-activation with the PDZ-binding motif. Furthermore, protein prenylation appears to be strongly related to oncogenesis and post-translational modification of growth-related oncoproteins such as Ras, Rac, and the Rho GTPase family . Among ahologies ,96. The t cancer ,97,98,99t cancer ,98. Furtt cancer . Interest cancer ,100, butt cancer . The antt cancer .Leishmania donovani, Leishmania major and Toxoplasma gondii), fungi , and bacteria  [Plasmodium falciparum [Toxoplasma gondii [Babesia microti [Mycobacterium tuberculosis [The use of MVA pathway inhibitors as antibiotics and antiviral agents was also explored in several reports. Among the organisms targeted, there were viruses , protozoa  ,104,105. aureus) ,104,105.lciparum ,107,108,a gondii , Babesia microti , or Mycorculosis . Furtherrculosis . Importarculosis ,113,114.The polyprenol salvage pathway plays a role in several conditions etiologically related to isoprenoid deficiencies. These deficiencies may occur as a side effect of pharmacological treatment, during aging, due to a genetic disorder or idiopathic reasons. For exemple, osteoporosis is a bone calcification deficiency that correlates with aging and is usually treated with biphosphponates . OsteoneStatins, used to manage dyslipidemias, also possess several side effects, including headache, dizziness, discomfort or weakness in skeletal muscles, constipation, diarrhea, and hepatotoxicity . SpecifiIt could be envisaged that FOH may promote dyslipidemias, as some studies demonstrated that this compound is converted into cholesterol in human cells due to the polyprenols salvage pathway . Howwww.orpha.net, last access: 21 October 2022) is caused by hypomorphic mutations, and the patients typically display fever episodes and hyperimmunoglobulinemia [MVK gene brings about mevalonic acidura , a severe form of the condition associated with short life expectancy and dysmorphic features. Since the major effects of these diseases are also related to defects from isoprenoids biosynthesis and protein prenylation, the dietary supplementation with polyprenols has been tested as a treatment for mevalonate kinase deficiency. Again, the administration of geraniol, FOH, and GGOH improved symptoms and inflammation markers both in in vitro and in vivo models [Mutations in the human mevalonate kinase gene are the cause of a short spectrum of related rare diseases. Mevalonate kinase deficiency (ORPHA code 309025; see ulinemia , while co models ,128,129.o models , as wello models ,58 or reo models . What iso models ,131. Whio models . Thus, tIn animals, both FOH and GGOH can be enzymatically synthetized or incorporated from the diet, producing diverse biological effects. Most effects are the consequence of the existence of a pathway for polyprenol salvage, composed by a polyprenol kinase and a polyprenyl-phosphate kinase. Once FOH and GGOH are pyrophosphorylated, they are incorporated into the protein prenylation process, as well as in other isoprenoid-dependent anabolic pathways, such as the synthesis of ubiquinone, cholesterol, and dolichol. This FOH/GGOH salvage pathway seems to be active in bacteria, animals, and plants as a mechanism to regulate or by-pass the isoprenoid biosynthetic pathway. However, to date, just a few enzymes have been identified, only in plants and cyanobacteria. Most likely, the study of this pathway has suffered from technical problems: first, these enzymes seem to present little sequence similarity to others with like functions, and no easy complementation assays are available. Furthermore, polyprenyl kinases are presumably membrane proteins and thus, are difficult to express heterologously. Enzyme activity and cellular polyprenol-distribution assays are cumbersome, since they usually require radio-labelling techniques, which are nowadays much in disuse. As a consequence, the enzymes responsible for this pathway in animals or any pathogen are still unidentified. However, considering the evidence, it seems patent that the identification of these enzymes could shed light on several infectious diseases, cancer, dyslipidemias, nutritional deficiencies, and the secondary effects of several drugs. Throughout this text, we aimed to present, in a simplified but comprehensive way, the main data on the FOH and GGOH salvage pathway, emphasizing its biomedical importance. Therefore, the main goal of this review was to raise awareness among the scientific community of the need for a better study of the polyprenol salvage pathway, and to discover the corresponding kinases in animals and pathogens.


Food frequency questionnaires (FFQ) is an easy and inexpensive tool that can be used to evaluate nutrient and dietary trends of groups and individuals. Few studies in the East Mediterranean region tailored FFQs to describe dietary intakes of older adults. The purpose of the study is therefore to assess the validity and reproducibility of a FFQ, designed for use with older adults living in a Mediterranean Arabic speaking country, Lebanon.The FFQ is composed of a list of 90 food items, commonly consumed by adults above 60 years of age. Validity of the FFQ was tested using the mean of two 24-hours dietary recalls (24HDR), and reproducibility, by repeating the questionnaire within a one-month period, along the second dietary recall. Our study included 42 and 76 participants, for the repoducibility and validity analysis respectively. Subjects were randomly selected from 2 of the 8 governorates in the country.FFQ reproducibility showed a mean relative difference of 1.03% without any significant difference between all paired components of nutrients. Intra class correlation (ICC) showed good and excellent reliability for caloric intake and all macronutrients, moderate to good reliability for all remaining nutrients, except for poly-unsaturated fatty acids, vitamins A, B12 and fibers. Correlation coefficients for all nutrients were fair to strong. Both administrations of the FFQ showed good internal validity. Validation of FFQ showed a mean relative difference between FFQ and mean 24HDR at 19.5%. Agreements between the 2 methods, for classifying individuals in the same or adjacent quartile, for nutrient intake and nutrient adequacy, were 80 and 78.2% respectively. Mean Kappa coefficient was 0.56 and energy-adjusted correlations were within the recommended values for all items except for vitamin A and B12. Adjusting for nutrient-dense food intake improved the agreement for theses 2 vitamins to 0.49 and 0.56, respectively.The proposed FFQ can be considered a valid tool to help describe nutrient intake of older individuals in an Arabic speaking Mediterranean country. It could serve for possible use in the East Mediterranean region for the evaluation of regular dietary intake of community-dwelling older adults.The online version contains supplementary material available at 10.1186/s12937-022-00788-8. Understanding the impact of dietary factors on the development of chronic diseases in elderly, is important for better prevention and treatment –6. ChoosFrequently used direct retrospective methods are diet records (DR), 24 hours dietary recalls (24HDR) and food frequency questionnaires (FFQ). They can be self-reported or interviewer- reported depending on the needs of the study. To reflect chronic and habitual intakes, it is recommended that DR and 24HDR tools be repeated over several days. These recommendations might be difficult to apply, expensive and time consuming particularly in the setting of lack of literacy and old age, especially in large scale population-based studies –9.FFQ is easier and less costly and can be administered to large cohorts. It usually can categorize and rank individuals by their usual frequency of food consumption. FFQ allows for comparison of food and nutrient intake across populations and countries with similar cultures and food availability , 10–13 aEach FFQ is constructed based on specific study objectives and target populations and needs to be validated to serve other groups and other study designs. For more reliable results, adaptation should therefore be made to the age-group targeted , 16. TheNon-institutionalized older adults in Lebanon are susceptible to a higher risk of malnutrition and insufficient dietary intakes due to inappropriate or inexistant retirement plans, limited access to health care services and social welfare programs, as well as inadequate living conditions , 18. ThiQuestionnaires used to evaluate food intake of older adults is scarce, particularly in the East Mediterranean region. In neighboring countries and in Lebanon, other questionnaires were used to estimate food and nutrient intake in adult population, but so far, no FFQ was validated exclusively among Lebanese older adults , 19–24.The aim of our study is therefore to validate a quantitative FFQ, specifically tailored to evaluate nutrient consumption of community dwelling Lebanese, 60 years of age and older, using the 24HDR as a reference method. This questionnaire could serve for possible use in Lebanon and other Arabic speaking Mediterranean countries as well.To study the validity of the FFQ, the mean of two 24HDR was used as a reference method, and to test the reproducibility of the FFQ, the same questionnaire was administered twice within a one-month period.From January 2017 till June 2017, recruitment of participants and data collection were carried out in collaboration with the Ministry of Social Affairs (MOSA) through the medico-social centers that serve low to middle class income families. As illustrated in Fig. To avoid inter-rater and reporting biases, participants were administered twice the FFQ and 24HDR by the same interviewer, and all filled questionnaires were reviewed by the principal investigator and field interviewers before data entry.As illustrated by Fig. n = 7 declining to do the second interview, and n = 1 for incomplete FFQ). A total of 13 out of 42 had their questionnaire answered by accompanying person, because of low cognitive test results. From an initial sample of 100 individuals included to test for the validity of the FFQ, and after a preliminary analysis, 24 participants were not included in the study: subjects with previously undeclared active cancer (n = 2), unaccompanied individuals with altered mental status and who did not comply with the study protocol (n = 9), those refusing to undergo the second interview (n = 7), those having extreme daily dietary intake  or > 4000 Kcal/day (n = 2)), as well as those having extreme restrictions and may not be representing a regular feeding pattern (interview was performed during fasting for more than 14 hours period) (n = 3). A total of 34 out of 76 had their questionnaires answered by accompanying person, because of low cognitive test results. Retention rates within a one-month interval for repeated questionnaires, for FFQ and 24HDR, were respectively 84 and 76%.For both analyses, the size of samples was set for an expected intra-class correlation (ICC) value of 0.75 and a minimum acceptable value of 0.5, with 95% confidence and a desired power of 80%. The number of participants for the reproducibility study was 42 out of 50 initially selected for this analysis , coffee, tea and other drinks, methods of preparation were also detailed, and specific ingredients were added to the report.Daily food consumption, reported by the FFQ was analyzed by the Nutrilog software  to extract daily nutrient intake. The United States Department of Agriculture/ Standard reference 28 (USDA/SR28) and the Canadian Nutrient File (CNF 2015) databases were used to analyze nutrient composition of simple foods, and branded foods. For composite traditional dishes and some locally consumed desserts, not found in the database, recipes from known local cookbooks and pastry chefs were entered to the database and used in the nutrient analysis. Data on food consumed were reported simultaneously in (g) and as portions. The nutrient intakes of the two 24HDR were also extracted using the same methods.To analyze the concordance between FFQ and mean 24HDR in classifying individuals in terms of nutrient adequacy, we calculated The Nutrient Adequacy ratio (NAR) of 17 selected nutrients, including vitamins A, D, E, K, C, B1, B2, B3, B6, B9, B12, and minerals such as calcium, phosphorus, magnesium, iron, selenium and zinc. The NAR was calculated by dividing the estimated nutrient intake of individuals by the age and sex-specific recommended dietary allowance (RDA) for these nutrients, according to the established dietary reference intake (DRI) recommendations  For all p < 0.05 was considered significant. To assess the internal consistency of the FFQ, after standardization of the variables, all items were computed in Cronbach’s alpha statistic. Alpha values between 0.7 and 0.8 indicate a good reliability, and a value higher than 0.9 would imply that all items on the questionnaire were very strongly related to each other and reliable for assessing the construct. To measure within-person variability, intra-class correlations (ICC) were calculated between the first and the second FFQ administration, using one-way random effects model, where people’s effects are random whereas interviewers performed under the same conditions. Values of r < 0.5 indicate a poor reliability, between 0.5 and 0.75 a moderate reliability, between 0.75 - 0.9 a good reliability, and greater than 0.9 excellent reliability [IBM-SPSS 20.0 was used for statistical analysis. All macronutrients, 10 vitamins and 5 minerals estimated intakes were reported and compared for the validity and the reproducibility studies respectively. For numerical variables, data was expressed as mean ± standard deviation and median (interquartile ranges), and as numbers and percentages, for categorical variables. Student t-test and chi-square test were used to compare numeric data and categorical data respectively. Normal distribution of calories and nutrients was assessed using Kolmogorov-Smirnov tests, and data was processed accordingly. Characteristics of the reproducibility and the validation study samples were compared using Mann-Whitney U test, for continuous data and Chi-square test for categorical data. Wilcoxon signed-ranks tests were used for comparisons of both related samples of food frequency questionnaires, and for the difference between FFQ and mean 24HDR. For normally distributed data, Pearson’s correlation coefficient was used to test the reproducibility (test-retest) of the FFQ and the relation between estimated macro and micronutrients between the FFQ and 24HRs. For data that was not normally distributed, Spearman’s correlation coefficient was used. Correlation coefficients and their respective 95% confidence intervals (CI) were calculated and iability . Energy To measure agreement of NAR estimation between the FFQ and the mean 24HDR methods, we calculated the percentage agreement between NAR estimated by FFQ and mean 24HDR by comparing classification in the same or adjacent quartile to distant quartiles, and through Kappa statistics. Concordance between the 2 NAR classifications was also measured using Spearman’s correlation coefficient.N = 76), had a mean age of 74.4 ± 6.9 years and a mean BMI of 30.3 ± 6.2 kg/m2. In this sample, 63.2% were women, 60.5% being married, and 84.2% living with their partners or relatives. The group of individuals included in the reproducibility/reliability study (N = 42) resembles the validity study sample, and characteristics of both did not differ statistically.Characteristics of the sample population are presented in Table The reproducibility study included 42 individuals, by repeating the FFQ questionnaire within a one-month interval. Median and mean intakes ± standard deviation (SD) of calories, macronutrients, and micronutrients of both FFQ1 and FFQ2 are reported in Table r = 0.3-0.5) was noted for MUFA, PUFA, omega-3 fatty acids (ω3 FA), omega 6 to omega 3 fatty acids ratio (ω6/ω3 ratio) and cholesterol intakes.To measure within- person variability, ICC and correlation coefficients  were calculated comparing the two administrations of the FFQ. Based on ICC, a good and excellent reliability was noted for caloric intake and most macronutrients. Remaining nutrients had a moderate to good reliability except for PUFA, vitamins A, B12 and fibers. Correlation coefficients for fibers, vitamin A and B12 showed a strong association between both administrations, as found for caloric intake, all macronutrients and micronutrients. Fair correlation (r = 0.82 (95% CI = 0.792-0.846) and r = 0.845 (95% CI = 0.77-0.905).Internal consistency, measured by Cronbach Alpha based on standardized items, was respectively 0.947 and 0.962 for the first and second administrations of the questionnaire, and their mean ICC, was respectively Caloric intake and intake of macronutrients and some relevant micronutrients estimated by the FFQ, were compared to the mean of both 24HDRs, allowing to evaluate the accuracy of the FFQ in our study sample and estimate its agreement with this chosen standard. Details of mean and median daily intake of nutrients and their correlations are shown in Table Interrater reliability between FFQ and 24HDR was evaluated thru percentage agreement and Kappa statistics for all listed nutrient intakes. Percentage agreement between the two tools, calculated by the ratio of classification in the same or adjacent quartile, to distant quartiles, placed older individuals in the same or adjacent quartiles, on average, in 80% of the sample population, with agreements ranging from 90.8% for sugars to 61.8% for vitamin B12. Based on Kappa coefficient, agreement between FFQ and 24HDR was, strong (> 0.61), for total calories, total fat, sugars, vitamins E, C, B1, B3 and calcium. Agreement was moderate (0.41-0.6) for all remaining nutrients except for vitamins A, B12 and fibers with poor agreement. Mean Kappa value was 0.56.Energy adjusted correlation between FFQ and mean 24HDR was performed by pairwise linear regression models and shown in Table We selected a group of nutrients to evaluate the bias between the nutrient mean differences and determine the agreement interval of the differences, between the FFQ and reference method we used. This agreement, performed thru Bland-Altman analyses, is represented in Fig. Table The present study showed that the proposed 90 food-items FFQ gives a good estimation of nutrient intake of older individuals in an Arabic-speaking Mediterranean country. The analysis showed that the FFQ has a good reproducibility and reasonable validity in relation to the mean of two 24HDR.The results obtained in our repeatability study showed that all pairs of intakes estimated by the 2 FFQ administrations showed no significant difference for most macronutrients and micronutrients. FFQ administrations showed excellent internal consistency. The ICC analysis of the FFQ showed good reliability. For most nutrients, these findings align well with those reported in the meta-analysis on the reproducibility of FFQ by Cui et al., where the median of ICCs for the elderly (> 50 years) was good, with ICC coefficients ranging from 0.482 to 0.866, lower than those for younger age-groups for 22 of 40 nutrients analyzed. . When coIn our validity study, the percentage difference between the two tools showed a mean percentage difference between the FFQ and the reference method of 19.45%, with FFQ overreporting nutrient intake. As often reported, when comparing the FFQ to the mean of 24HDR, FFQ tends to overestimate nutrient intake for almost all nutrients , 42.Agreement between FFQ and 24HDR was measured by first ranking participants’ dietary intake in quartiles, then assessing the percentage agreement between the same or adjacent quartiles and calculating Cohen’s Kappa coefficient and correlation coefficients. We were able to show that the used instruments are highly concordant. For most of nutrients, our FFQ ranked correctly around 80 and 78% of individuals, in the same or adjacent quartile as the mean 24HDR, whether in classifying nutrient intake or nutrient adequacy respectively. Moreover, Kappa coefficients detected moderate to good agreement between nutrient estimation and adequacy classification of participants between FFQ and mean 24HDR. Our results are concordant with previous reports where it was established that 50% of subjects should be correctly classified, and weighted kappa values should be above 0.4 for nutrients of interest , 44.Our findings showed that FFQ overestimated intakes when compared to 24HDR, and correlation improved when adjustment for calories is made. Energy-adjusted nutrient intake estimated by FFQ compared to mean 24HDR, indicated correlation coefficients higher than 0.5 for all macronutrients and most micronutrients. A fair correlation only occurred with vitamins A, and B12. As reported by other studies, vitamin A intake is usually difficult to assess by FFQ, probably because of double counting of items, uneven distribution of vitamin A across food items, seasonal changes in dietary habits, social desirability bias, and overreporting of healthy nutrient-dense foods , 45, 46.The main strength of our study was the consideration of age-related cognitive decline while assessing the validity and reproducibility of the FFQ, performed in a homogeneous older population. Previous reports in elderly population found errors in evaluating total energy intake related to cognitive ability, with an inverse association between reported calories and cognitive functions , 48. In To our knowledge, no previous validated questionnaire was developed to specifically describe and evaluate dietary habits of older individuals in the region.This study has few limitations. To assess intake over 1 year, 4 days recall (1 day per season) are recommended to rule out seasonality bias . Our choSpecial consideration should be given for seasonality that might affect older adults reporting on their food intake. We suggest repeating the FFQ bi-annually to represent more specifically habitual intake. A self-reported version of the FFQ questionnaire could be tested for validation to allow a wider use of the questionnaire among literate elderly participants in the future.In this study we were able to test the validity and the reliability of a culture specific FFQ adapted to the elderly population in the Middle East Mediterranean region as compared to mean of two 24HDR and confirm that the FFQ remains one of the most suitable and inexpensive choices to describe food intake of elderly people of low economic status .Evaluation of food intake in elderly individuals is quiet challenging, and few countries in the Middle East developed and validated questionnaires in people aged 60 years and more. Our questionnaire showed satisfactory reliability and validity and could be suggested for a broader application as a suitable tool to estimate and evaluate dietary habits and nutrient intakes of older individuals living in Lebanon and in the East Mediterranean Arabic speaking countries.Additional file 1.


Houttuynia cordata (H. cordata) is the most common herb as a food and traditional Chinese medicine. Currently, studies on its toxicity have mainly focused on hepatotoxicity. However, its potential embryotoxicity by long-term exposure is often overlooked. Objective: To investigate the effects of H. cordata on embryonic development and its toxicity mechanism by combining network pharmacology, molecular docking, and in vitro experimental methods. Methods: The effects of H. cordata on embryos were evaluated. Zebrafish embryos and embryoid bodies were administered to observe the effects of H. cordata on embryonic development. Based on network pharmacological analysis, it was found that the main active agents producing toxicity in H. cordata were oleanolic acid, lignan, and aristolactam AII. H. cordata can affect PI3K-Akt, MAPK, and Ras signaling pathways by regulating targets, such as AKT1, EGFR, CASP3, and IGF-1. RT-PCR and immunohistochemistry results showed that the expression of AKT1 and PI3K in the embryoid body was significantly reduced after drug administration (p < 0.05). Conclusions: The results of network pharmacology and in vitro experiments suggest that H. cordata may affect embryonic development by influencing the PI3K-Akt signaling pathway. People generally believe that medicinal and edible plants have low toxicity and side effects and pay little attention to the rational use of a medicinal and edible plant with long-term exposure , leadinghrotoxic . When thonfusion . With thonfusion , it is iHouttuynia cordata (H. cordata) is a perennial herb in the Saururaceae family [H. cordata was first recorded in Ming Yi Bie Lu more than 1500 years ago. It is widely distributed in China, Korea, Japan, India, and other Asian regions, and is important in China [H. cordata has anti-mutagenic [H. cordata is also widely consumed as a healthy vegetable in East Asia [H. cordata was tested in a single dose and long-term toxicity in rats, and the results showed toxicity to rat liver and kidney [H. cordata and the 95% ethanol extract of the rhizoma H. cordata had certain acute toxicity and embryonic development toxicity to zebrafish adults and embryos. H. cordata is often used as a way to eat stews and boiled dishes, and the method of water extract is closest to daily consumption [H. cordata has great practical significance. However, the embryo safety of water extract H. cordata remains unknown.e family . H. cordin China . Currentutagenic , anti-cautagenic , anti-obutagenic , hepatoputagenic , anti-viutagenic , anti-bautagenic , anti-inutagenic , anti-alutagenic , anti-leutagenic . H. cordast Asia . For exad kidney . Furthersumption . The stuH. cordata more sensitively. We then used a network pharmacology method to unravel the mode of action of H. cordata in terms of embryotoxicity. Simultaneous validation of core targets was performed using network pharmacology and molecular docking. This study will facilitate the safety assessment of H. cordata and provide information for the clinical use of H. cordata. In 2007, the British pharmacologist Hopkins formulated the idea of network pharmacology . NetworkH. cordata on embryonic mortality in zebrafish, Zebrafish embryos at 0 hpf were exposed to 2, 2.5, 3, 3.5, and 4 mg/mL of each tested compound until 120 hpf. The first row of H. cordata drugs. At 96 and 124 hpf, zebrafish were administered with 3.5 and 4 mg/mL of H. cordata solution, died, and had curved spines. The red arrows in For the assessment of the effect of H. cordata on the survival percentage of 24, 48, 72, 96, and 120 hpf is shown in H. cordata markedly decreased the survival rate of zebrafish embryos. At 120 hpf, all embryos of 3 mg/mL H. cordata zebrafish died. The LC50 of H. cordata at 120 hpf were 2052 μg/mL.The influence of H. cordata markedly decreases the hatching rate of zebrafish embryos , small liver, and abnormal intestine  were evaluated over 4 days. Unfavorable morphogenetic effects were observed when EBs were exposed to concentrations of 150 μg/mL or more. EBs were smaller  than the control when exposed to 150 μg/mL. When treated at 350 μg/mL, cells did not survive forming EBs. To further refine the concentrate–response relationship, EBs were exposed to 150, 250, and 350 μg/mL of H. cordata  analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis are essential sub-analyses in network pharmacology to analyze the function of genes and the associated enriched pathways. Enrichment analysis was performed by GO and KEGG. 126 cross-targets were entered into the Metascape system for GO enrichment analysis and pathway enrichment analysis. The biological processes (BP), molecular functions (MF), and cellular components (CC) associated with GO results revealed the functions of these underlying targets. Altogether, 2766 significantly enriched GO entries were derived from Metascape, including 1642 BP, 723 MF, and 401 CC entries, based on activity . Among 4e picked .H. cordata, in this part, a systematic and comprehensive bioinformatics network of herbal medicine–active compound–central target–pathway–embryotoxicity network (C-E-H-P-E) was constructed by 51 active compounds, 126 potential gene targets, and 20 KEGG signaling pathways. The complex network consisted of 211 nodes and 2572 edges, showing complex interactions between various compounds, targets, and pathways. In the bioinformatics network, purple represents herb; blue indicates bioactive compounds from H. cordata; yellow stand for the target genes; green represents signal pathway; red is a disease. One gene target corresponds to multiple compounds, and one potential active compound corresponds to multiple targets. It could be seen that the mechanism of H. cordata’s embryonic toxicity is characterized by several components, multiple targets, and various mechanisms. The details of the Chinese medicines-effective compounds-hub targets-pathways embryotoxicity network are shown in H. cordata were strongly associated with signaling pathways, as shown in To further illuminate the embryotoxic mechanism of action of H. cordata were screened for molecular docking. Before performing molecular docking of H. cordata components, we first verified the reliability of the molecular docking process. Since the crystal structure of the AKT1 (4EJN) target protein contains the original ligand of 0R4. we extracted the ligand molecule from the complex and then docked it into the binding pocket of the complex using LibDock. The RMSD of the docked ligand from the ligand conformation in the original crystal structure was calculated to be 1.3447, which is less than 2.0, indicating that the docking procedure has good accuracy for this target. Subsequently, we performed molecular docking with three active ingredients, luteolin, Aristolochia lactam AII, and oleanolic acid, as ligands and key target proteins as receptors. The docking results showed that the docking scores of all three active compounds with the core target AKT1 were greater than 100, indicating a relatively stable binding between ligand and receptor  value can visualize the interaction and stability of the docking model. Here, we constructed AKT1-based protein receptors, which are the core targets of PPI. Based on network pharmacology, three highly active compounds from receptor .H. cordata active compounds mentioned above. A high similarity was observed between the active compounds and the original ligands of the proteins, which occupy almost the same active site. The binding pattern between oleanolic acid and AKT1 is shown in H. cordata significantly inhibited the expression of PI3K AKT1 gene , which contains endodermal, mesodermal, and exodermal tissues, and can simulate the dialogue and mutual induction process among multiple layers in vivo. When EBs were treated with H. cordata at 150 μg/mL and higher doses, adverse morphogenetic effects were observed. The second day of EBs formation corresponds to the onset of embryonic development of implantation and protointestinal formation [H. cordata was significantly toxic to different cell lines from protointestinal embryo formation to differentiation.First, in an in vivo experiment, we investigated that the mortality of zebrafish embryos is enhanced in a concentration and a temporal-dependent way. The LCormation . On day H. cordata are focused on hepatic and renal toxicity, and there are few reports on embryotoxicity due to insufficient model test sensitivity. We used zebrafish and embryoid bodies assay to verify embryotoxicity, which was more sensitive than traditional animal experiments. It can be determined that H. cordata has a potential embryotoxic risk. Next, the possible mechanism of H. cordata embryological toxicity was systematically studied by network pharmacology and molecular docking analysis. The composition-target-toxicity network of H. cordata on embryotoxicity was analyzed and constructed. 52 main active ingredients of H. cordata and 126 common targets of embryoid bodies, including oleanolic acid, Luteolin, and Aristolochia lactam AII. Studies have shown that oleanolic acid can cause liver damage when taken in small doses over a long period or in large doses over a short time, Cholestasis is the main feature of liver injury [H. cordata play an important role in embryotoxicityMost of the reports on the toxicity of r injury . Liver dr injury . Thus, cH. cordata acts on several targets through various signaling pathways, mainly AKT1, EGFR, IGF-1, and CASP3, and that these potential core target genes may play a crucial role in embryotoxicity. EGFR is a specific receptor of the tyrosine kinase receptor family that acts on its placental target cells (trophoblast cells). Changes in the biological activity of EGFR can affect fetal development [Using integrated network analysis, we found that elopment . EGFR inelopment . At the elopment . The proelopment . The serelopment . Studieselopment . Insulinelopment . IGF-1 pelopment . It was elopment . In concH. cordata may produce embryotoxicity through the PI3K-Akt signaling pathway, and studies suggest that PI3K-Akt is an important trophoblast regulatory signal in humans [Within our research, KEGG enrichment analysis revealed 126 common targets mainly associated with PI3K-Akt, Ras, and MAPK signaling pathways. n humans . Embryonn humans . Our datH. cordata produces embryotoxicity.Our study further analyzed the BP from GO to identify the relationship between underlying targets and embryotoxicity. Based on what we have analyzed, the most abundant BP were cellular response to hormone stimulus, cellular response to organic cyclic compound, and cellular response to nitrogen compound. Nitrogen acquisition and utilization are critical in embryonic development . This prH. cordata can bind more stably to core targets of embryotoxicity. Further, immunohistochemistry and qRT-PCR experiments were performed on the highest-scoring target AKT1 and PI3K, and the results showed that Ichthyophonus had an inhibitory effect on AKT1 and PI3K. Our data suggest that PI3K-Akt may be the most central signaling pathway for the production of embryotoxicity in H. cordata.Molecular docking is the most commonly used method to assess component–target interactions. Luteolin, Aristolochia lactam AII, and oleanolic acid bind most stably to AKT1, both with docking fractions < 100. AKT1, EGFR, CASP3, and IGF-1 showed a strong association with all three core components with docking scores < 80. Major components of H. cordata on the development of zebrafish embryos and EBs with iPSCs grown in three dimensions and zebrafish embryos as the model organism. The results showed that the effect of H. cordata embryos was evidenced by the reduced diameter of EBs, reduced hatchability, mortality, and teratogenicity of zebrafish embryos, and target organ toxicity. Network pharmacology identified 51 key active compounds and 126 genes targeted by embryotoxicity in H. cordata. By integration of network pharmacology, three core targets from a complex network  were predicted as key targets for embryo production in H. cordata. In addition, we discovered that the PI3K-Akt signaling pathway may be central in generating the embryotoxic system. The results of molecular docking further showed that H. cordata showed a very good affinity for the core targets, and the immunohistochemical and qRT-PCR results demonstrated the inhibition of AKT1 and PI3K by H. cordata. In summary, this research offers a clear basis for the generation of embryotoxicity in H. cordata and presents a comprehensive and novel approach to the search for active ingredients, nuclear target genes, and underlying mechanisms of herbal medicines. Pregnant women should eat with caution or watch the amount they consume.In this study, we investigated the effects of H. cordata (XEB181) from Lishui, Zhejiang Province was obtained from Huamiao Pharmaceutical Co., Ltd. , and the drug was extracted with water (5 drinks g/extract g) from the Chinese Academy of Traditional Chinese Medicine. The quality of H. cordata was controlled by efficient liquid chromatography (HPLC) (H. cordata and treated with ultrasonic waves for 10 min before use.y (HPLC) . A mothe3), provided by the fish farming center of Huante Biotechnology Company Limited. The laboratory animal permit number is SYXK (Zhejiang) 2012-0171, and the raising management satisfies the international AAALAC requirements for approval (certification number: 001458). For breeding, males and females are separated in a spawning box (having a glass partition) in a proportion of 1:1. The males and females are then mixed and spawned during the early morning hours. Normally developing embryos are captured for lab studies [AB–Adult feral type zebrafish were fed in fish farming water at 28 °C  were determined by preliminary tests. Embryos were randomly collected and transferred to 6-well plates after 0 h post-fertilization (hpf), with 30 embryos and 3 mL of H. cordata solution deposited in per well. Each concentration and control was replicated 3 times. All test solutions were changed every 24 h. Dead embryos were cleaned up in and timely recorded.The embryo acute toxicity test was conducted following the methods prescribed by the Organization for Economic Cooperation and Development (OECD) ,39. ExpeEach treatment group was watched for developmental toxicity in the exposure period with light microscopy . Calculation of zebrafish mortality at 24, 48, 72, 96, 120 hpf, and 72 hpf hatch . At 24, ® Human Pluripotent stem cell Digestive Fluid  and seeded onto ultra-low adhesion U-bottom 96-well plates at a density of 1 × 104 cells/mL in 100 μL EBs differentiation medium (ESC medium without LIF). Embryoid bodies were obtained the next day [Human-induced pluripotent stem cells (hiPSCs) were purchased from Beijing Saibei Biotechnology Company, placed on 0.1% gelatin -coated tissue culture plates, and cells were maintained in the mTeSRTM1 complete medium . For embryoid body (EBs) generation, ihPSCs were dissociated to single cells with PSCeasynext day .H. cordata  were determined with the preliminary test. A blank group and a positive control group of mifepristone were also set. Select EBs with uniform appearance and size for culture, and start adding medicine two days later. EBs were cultured for up to 4 days in an incubator with 5% CO2 at 37 °C in humidified air. Media were replaced every other day. The survival and integrity of EBs were monitored daily, and the diameter of EBs was measured using a live cell imager. Chemical treatment was considered to have an unfavorable morphogenetic influence when EBs displayed a mean relative diameter that was more than 18% lower than that of the control group [The experimental concentrations of ol group ,43. Eachp < 0.05.The results were calculated using GraphPad Prism 8.0 software . The measured data were statistically analyzed using one-way ANOVA and the data were analyzed by one-way ANOVA and chi-square test. The results were analyzed using the mean ± standard deviation (x ± s) The results were expressed as mean ± standard deviation (x ± s), and the differences were considered statistically significant at https://tcmspw.com/tcmsp.php, accessed on 22 June 2022), we searched for the effective ingredients of herbal medicine and added them to the published literature. We carried out preliminary filtering of active ingredients to acquire the active compounds and their protein targets, where oral bioavailability (OB) ≥ 30% and drug similarity (DL) ≥ 0.18% were set as criteria. The SwissTarget Prediction platform  was then used to predict potential gene targets. Following the screening, the UniProt database  was also used to standardize the targets of the proteins in which the compounds act to obtain more comprehensive target information for normalizing protein target information [Through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  and OMIM database  for gene targets associated with embryotoxicity using the keyword “embryotoxicity”. Targets related to embryotoxicity were acquired by combining targets from the two databases and deleting duplicates. These co-targets were then filtered using Venny 2.1  and classified as possible targets for H. cordata in embryotoxicity [We mined the GeneCards database . The organism type was assigned as “Homo sapiens” and the confidence score of the degree of correlation was set to ≥0.4. After hiding the unlinked nodes, the outcome is exported in Tab-separated value (TSV) format. Then, Cytoscape v3.7.2 was employed to visually process the PPI data to filter the kernel targets with the variables of betweenness centrality (BC), proximity centrality (CC), and degree centrality (DC)[PPI networks were structured by presenting common disease–drug targets to a search vehicle for the retrieval of interacting genes .H. cordata were imported into the Metascape system . We selected “Homo sapiens” as the species and a significance level was set at p < 0.01 to complete GO functional analysis and KEGG pathway analysis. GO enrichment was performed to cover three aspects of biology: cellular components, molecular functions, and biological processes. KEGG enrichment analysis can also suggest the biological mechanisms of drug action in humans and the pathways involved in their regulation [Previously gained embryotoxicity targets of gulation .H. cordata and Embryo toxicity [Network visualization software Cytoscape 3.7.2 was utilized to build the network, a network visualization software to intuitively display the compound–target–pathway relationship between toxicity .http://www.rcsb.org/, accessed on 20 July 2022), after which the original ligand is abstracted and then docked into the binding pocket of the complex and the root-mean-square deviation (RMSD) of the docked ligand conformation from the ligand conformation in the original crystal structure is calculated. The method is generally considered reliable when the RMSD is less than or equal to 2.0.In the current study, we selected the three active ingredients predicted by web pharmacology as ligand compounds and selected AKT1, which scored the highest in the web pharmacology search filter, as the receptor protein and downloaded the crystal structure of the protein. Molecular docking was performed using Discovery Studio, and the active site was determined using the ligand expansion method, i.e., the location of the ligand was taken as the center and then extended outward to a certain range, generally with a radius of 9 Å, and the receptor residues within this range constituted the relevant active site. Before the molecular docking of the three active ingredients, the accuracy and reliability of the docking process should be evaluated. The target protein complex containing the original ligand is first obtained from the RCSB PDB database  and phosphate-PI3K  overnight at 4 °C while PBS was added dropwise to each group of samples as a negative control. Subsequently, sections were incubated with goat anti-rabbit enzyme-labeled secondary antibody for 2 h at room temperature and diaminobenzidine for visualization. Finally, these sections were observed and analyzed using a Cytation 5 microscope .Well-shaped EBs were selected and fixed in 4% paraformaldehyde-phosphate buffer salt solution fixative for more than 24 h. EBs were sedimented in sucrose solution, embedded in OCT, and sliced with a frozen section machine  at a thickness of approximately 12–15 μm. Subsequent frozen sections were fixed and antigenically repaired with EDTA antigen repair, endogenous peroxidase was blocked with 1% H−ΔΔCTmethod.Total sample RNA was extracted using Trizolszol up  and reverse transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA synthesis SuperMix  according to the manufacturer’s guidelines, RT-PCR was performed on an ABI Step One system  using the TransStrart Green QpcrSuperMix kit . The specific primers used for RT-PCR are as follows. PI3K , AKT1 , GAPDH . GAPDH was used as a normalization control. The relative RNA expression of each gene was analyzed by the 2


Precision medicine aims to address the demand for precise therapy at the gene and pathway levels. We aimed to design software to allow precise treatment of osteoporosis (OP) with Chinese medicines (CMs) at the gene and pathway levels.PubMed, EMBASE, Cochrane Library, China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database (VIP database), and the Wanfang database were searched to identify studies treating osteoporosis with CMs. The TCMSP was used to identify bioactive ingredients and related genes for each CM. Gene expression omnibus (GEO) database and the limma package were used to identify differentially expressed genes in osteoporosis. Perl software was used to identify the shared genes between the bioactive components in CM and osteoporosis. R packages and bioconductor packages were used to define the target relationship between shared genes and their related pathways. Third-party Python libraries were used to write program codes. Pyinstaller library was used to create an executable program file.Data mining: a total of 164 CMs were included, but Drynariae Rhizoma (gusuibu) was used to present this process. We obtained 44 precise relationships among the bioactive ingredients of Drynariae Rhizoma, shared genes, and pathways. Python programming: we developed the software to show the precise relationship among bioactive ingredients, shared genes, and pathways for each CM, including Drynariae Rhizoma.This study could increase the precision of CM, and could provide a valuable and convenient software for searching precise relationships among bioactive ingredients, shared genes, and pathways.The online version contains supplementary material available at 10.1186/s13020-022-00596-6. Osteoporosis, a systemic skeletal disease, is defined by an overall deterioration of bone mass and bone microstructure , consequTraditional Chinese medicine (TCM) has become increasingly popular because of its effectiveness and fewer side effects. Natural Chinese medicine, with its effects on the growth and development of skeletal tissue , 28, hasPrecision medicine, a movement in clinical practice, aims to develop treatments that specifically address the demand for precise therapy at the gene and pathway levels . In the In this context, we aimed to obtain ‘precision TCM’ to facilitate the precise treatment of osteoporosis with CMs at the gene and pathway levels. As precision medicine moves forward, new strategies require carriers to express them . The PytSix databases including, PubMed, EMBASE, Cochrane library, China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database (VIP database), and Wanfang database were searched from their inception till August 18th, 2021. All studies published in English and Chinese were searched. The detailed search strategy for PubMed is shown in Appendix A.The retrieved literature from electronic databases was imported into NoteExpress to delete duplicates. Two authors (Tang and Wang) independently screened the titles, abstracts, and full texts of the remaining studies to identify eligible studies according to the inclusion and exclusion criteria. The inclusion and exclusion criteria for original studies is as follows: (1) Patients with osteoporosis were included. (2) Interventions involving Chinese medicines were included. (3) Any study design was included. (4) Literatures unabling to obtain Chinese medicines were excluded. The same authors independently extracted Chinese medicines from the eligible studies. Any disagreement was submitted to a third author (Jinyu Li) and resolved by his judgment.http://tcmspw.com/tcmsp.php) was used to extract all components of each CM included in this manuscript. The processes of absorption, distribution, metabolism, or elimination can affect pharmacodynamics and cause changes in drug bioavailability. Oral bioavailability was calculated using OBioavail1.1  Affymetrix Human Genome U133 Plus 2.0 Array platform, which included five osteoporosis samples and five non-osteoporotic samples.The Gene expression omnibus (GEO) |> 1 and P < 0.05. To visualize the differentially expressed genes, the ggplot2 and pheatmap packages were used to draw volcano maps and heat maps.Bioactive ingredients of CMs shared common genes with osteoporosis. Perl software was used to acquire the shared genes.In order to explore the pathways of shared genes between bioactive ingredients of each CM and osteoporosis, we installed the R packages , and to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, bioconductor packages  in the R software were installed. The species was set to “hsa,” and the filter values  were set to 0.05. Subsequently, we manually summarized the precise relationships among bioactive ingredients, shared genes, and pathways.We used the Python programming language to write software source codes in Pycharm Community Edition 2021.2, for which we called six third-party libraries of Python . We used pandas and openpyxl libraries to locate, read and retrieve data files; and tkinter, tkinter.ttk and ttkthemes libraries to write the overall interactive interface, to arrange various interactive elements such as input and output; further, we used tkinter.messagebox library combined with Python 3 basic syntax to create a trial-and-error mechanism. Finally, we generated a runnable Python file and used the pyinstaller library to package the Python file into an executable program.We previously performed cell experiments, which were published in the Chinese Journal of Tissue Engineering Research in 2020  to confiA preliminary search of the electronic databases retrieved 8866 articles. A total of 5346 articles remained after the deletion of duplicates using the NoteExpress software. Among these, 2688 articles were excluded based on the title, abstract, and full-text reading. A total of 2658 articles were left to extract data on CMs. We extracted 418 CMs from the eligible literature, of which 246 were unavailable in the TCMSP database. Therefore, a total of 172 CMs were included in our study , Borneolum Syntheticum (bingpian), Aconiti Radix (chuanwu), Rhizoma Dioscoreae Nipponicae (chuanshanlong), Dichroae Radix (changshan), Zanthoxylum nitidum (liangmianzhen), Trichosanthis Radix (tianhuafen), and Tetrapanacis Medulla (tongcao). Therefore, 164 CMs were used for the operations mentioned in the Methods section |> 1, a total of 2789 genes were acquired (1465 upregulated genes and 1324 downregulated genes). As shown by the gene volcano and heat maps  as an example to present the functions of the executable program file software as follows Fig. .Fig. 8ThOur software showed that the flavone Rhizoma Drynariae (gusuibu in lowercase pinyin) might treat osteoporosis via the Wnt signaling pathway Fig. . Li et aMouse MC3T3-E1 osteoblast line was provided from Peking Union Medical College, Beijing, China; Rhizoma Drynariae total flavonoids were provided from Beijing Qihuang Pharmaceutical Co., Ltd.① Normal group.② DKK1 group: Wnt pathway inhibitor DKK1 (0.1 mg/L) blocked the Wnt/β-catenin signaling pathway;③ DKK1 + transforming growth factor β (10 μg/L) group;④ DKK1 + total flavonoids of Rhizoma Drynariae (100 mg/L) group;⑤ DKK1 + total flavonoids of Rhizoma Drynariae (250 mg/L) group;The cells were harvested at both 24 and 48 h of treatment.Compared with the DKK1 group, the DKK1 + transforming growth factor β group, and the DKK1 + total flavonoids of Rhizoma Drynariae  groups had a higher mRNA expression of β-catenin, RUNX2 and Cyclin D1 (P < 0.05), and had a lower mRNA expression of GSK-3β (P < 0.05) after 24 h of treatment , might treat osteoporosis via the MAPK signaling pathway Fig. . Wu et aOsteoporosis, the most common chronic metabolic bone disease, is characterized by low bone mass and microarchitectural deterioration of bone tissue. Osteoporosis can enhance bone fragility and increase the risk of fractures . It has TCM can promote bone formation via osteogenesis of MSCs and osteoblasts . In KorePrecision medicine aims to maximize the therapeutic effectiveness by considering individual differences in genes, environment, and lifestyle . We are As precision medicine moves forward, new strategies require carriers to express them. In this study, we successfully created an executable program file to achieve precise treatment of osteoporosis using CMs at the gene and pathway levels, and supported the reliability and facticity of our software by our experimental data  and seveOur study showed that the combination of data mining and Python programming could be applied to design software to achieve precise treatment of osteoporosis with CMs at the gene and pathway levels. The results of our study demonstrated that to some extent, this executable program file may achieve precision treatment of CMs for osteoporosis, and may unveil the biochemical basis and underlying mechanisms of CMs for treating osteoporosis. Our previously published study  and seveAdditional file 1. The contents of data mining.Additional file 2. The executable program file software.