Glycine max. The study aimed to clarify not only the impact of over-expressing five terpene synthesis genes isolated from G. max and Salvia guaranitica on soybean nodulation signaling pathway, but also on the strigolactones pathway. The obtained results revealed that the over expression of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS genes enhanced the root nodule numbers, fresh weight of nodules, root, and root length. Moreover, the terpene content in the transgenic G. max hairy roots was estimated. The results explored that the monoterpenes, sesquiterpenes and diterpenes were significantly increased in transgenic soybean hairy roots in comparison with the control. Our results indicate the potential effects of terpenoids and terpene synthesis genes on soybean root growth and nodulation. The study provides novel insights for understanding the epistatic relationship between terpenoids, root development, and nodulation in soybean.Root nodule formation in many leguminous plants is known to be affected by endogen ous and exogenous factors that affect formation, development, and longevity of nodules in roots. Therefore, it is important to understand the role of the genes which are involved in the regulation of the nodulation signaling pathway. This study aimed to investigate the effect of terpenoids and terpene biosynthesis genes on root nodule formation in  Glycine max) is considered one of the oldest polyploidy (pa leopolyploid) plants and one of the most domesticated food crops in the world; it is expected to contribute to sustainable agriculture through its ability for symbiotic nitrogen fixation . Mor. MorGmFD plastid . These ipothesis A–H. The pothesis A–H. TerpGmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS genes enhanced terpene amassing in transgenic soybean hairy roots . Moreover, certain terpenes cause plant development and growth hence considered as primary metabolites rather than secondary metabolites [https://www.genome.jp/pathway/map00830+C02094, https://www.genome.jp/pathway/map00906+C02094, https://www.genome.jp/pathway/map01060+C02094, https://www.genome.jp/pathway/map01062+C02094, https://www.genome.jp/pathway/map01070+C02094, https://www.genome.jp/pathway/map01240+C02094, https://www.genome.jp/pathway/map04977+C02094, accessed on 28 July 2022). Apocarotenoids also include many phytohormones with important functions in plant–environment interactions such as abscisic acid (ABA) and strigolactones (SL) [https://www.genome.jp/pathway/map01070+C04691, accessed on 25 March 2021) were reported to affect plant growth, nodule formation, and interaction with other microbial communities [SoCINS, SoNEOD, SoSABS, SoLINS, SoGPS and SoTPS6 from S. officinalis affect nodule and root development in soybean transgenic roots, most likely by modulating terpene accumulation [GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS in transgenic soybean hairy roots has comparable effects on terpene accumulation and nodule formation.Remarkably, the overexpression of ry roots A,B. For  control . Moreoveabolites  , and assessing the root growth characters and nodulation as has been affected by the transgenic. Transgenic cultivated soybean overexpressing GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS presented meaningful changes in root development, nodulation, and the expression levels of nodulation signaling and SL biosynthesis genes. In silico tools and the putative expression analysis were employed to predict GmFDPS, GmGGPPS, SgGPS, SgFPPS,and SgLINS functions in root and nodule development. Our data affirm that the GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS promote root development and nodulation signaling by activating nodulation signaling and SL synthetic genes. As a result, this study provides a clearer vision of the function of the GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS in root development and nodulation, in conjunction with nodulation signaling and SL biosynthetic genes that are critical for legume nodulation production.In summary, this study focuses on cloning


Shengjing capsule (SJC) is a traditional Chinese medicine (TCM) and has gained widespread clinical application for the treatment of male infertility (MI). However, the pharmacological mechanism of SJC against MI remains vague to date.The active ingredients of SJC and their targets were identified from the database, and MI-related genes were retrieved from several databases. Protein–protein interaction (PPI) data were obtained to construct the PPI networks. The candidate targets of SJC against MI were identified through topological analysis of the PPI network. Functional enrichment analysis of candidate targets was performed, and the key target genes were identified from the gene-pathway network.RPS6, MAPK1, MAPK3, MDM2, and DDX5. Pathway enrichment analysis showed that SJC had the potential to impact multiple biological pathways, such as cancer-related pathways, viral/bacterial infection-related pathways, and signal transduction-related pathways.We identified 154 active ingredients and 314 human targets of SJC, as well as 564 MI-related genes. Eight pharmacological network diagrams illustrating the interactions among herbs, active ingredients, targets, and pathways, were constructed. The four dominating network maps included a compound-target network of SJC, a compound-anti-MI targets network, a candidate targets PPI network, a pathway-gene network, and a drug-key compounds-hub targets-pathways network. Systematic analysis indicated that the targets of SJC in the treatment of MI mainly involved Our results preliminarily revealed the pharmacological basis and molecular mechanism SJC in treating MI, but further experimental research is required to verify these findings.The online version contains supplementary material available at 10.1186/s12906-022-03774-z. Infertility, a disorder of the reproduction system, is characterized by the failure of a couple to achieve a clinical pregnancy after at least one year of unprotected and regular sexual coition , 2. MalePanax ginseng C. A. Meyer), LURONG (Cornu Cervi Pantotrichum), BAQIA (Smilax china L.), GOUQIZI (Lycium chinense Miller), HUANGQI  Bunge.), JINYINGZI (Rosae Laevigatae Fructus), YINYANGHUO (Epimedium brevicornu Maxim.), FUPENZI (Rubus idaeus L.), HUANGJING , XIANMAO (Curculigo orchioides Gaertn.), TUSIZI (Cuscuta chinensis Lam.), and BUGUZHI , has been wildly employed to treat male infertility in China. It was confirmed to improve oligozoospermia by enhancing spermatogenesis ability [In China, traditional Chinese medicine (TCM) has been wildly used in the treatment of male infertility for more than 2000 years with satisfactory results. Clinical studies on male infertility treated with TCM demonstrated the function of TCM to improve the quality of sperm and pregnancy rate of male interfile patients –18. In aNetwork pharmacology is a novel tool for discovering the mechanism of novel medicines and herbal medicines , 22. In http://lsp.nwu.edu.cn/tcmsp.php) and the Chinese Academy of Sciences chemistry database . The active compounds were identified if the chemical constituents meet the following criteria: oral bioavailability (OB) ≥ 40% and drug-likeness (DL) ≥ 0.2 [https://www.drugbank.ca/) [We mined the chemical constituents of SJC from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform   ≥ 0.2 . The tarank.ca/) .https://www.pharmgkb.org/), DisGeNET (https://www.disgenet.org/search), and GeneCards (https://www.genecards.org/).MI-related genes were obtained from the following 3 existing resources in March 2020 using “male infertility” as searching keywords: Comparative Toxicogenomics Database  [Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery   online tA total of 154 compounds in SJC were obtained from databases, including 7 in BAQIA, 44 in GOUQIZI, 10 in HUANGQI, 7 in JINYINGZI, 23 in YINYANGHUO, 7 in FUPENZI, 12 in HUANGJING, 7 in XIANMAO, 11 in TUSIZI, 22 in RENSHEN, 2 in LURONG, and 2 in BUGUZHI. Eventually, 123 active compounds were identified after removing the duplications, and 97 of the 123 active compounds had human targets   is crucial for the development and maturation of meiotic and postmeiotic germ cells in seminiferous tubes because it provided a unique microenvironment for these processes. [RPS6 participates in many pathways, including the mTOR and MAPK pathways. It has been revealed that RPS6 regulates the BTB dynamics spermatogenetic function in the testis [RPS6 in recurrent pregnancy loss (RPL) patients was significantly lower than in healthy control [RPS6 might contribute to MI and RPS6 can be a potential target for the treatment of MI. Mitogen-activated protein kinases (MAPKs) play a crucial role in the regulation of spermatogenesis and spermatozoa functions [MAPK1 and MAPK3 were also recognized as key target genes of SJC for male infertility due to their higher BC. A recent study found that DDX5 is expressed by spermatogonia and plays essential transcriptional and post-transcriptional roles in the maintenance and function of spermatogonia [TP53 has been confirmed to mediate the spontaneous testicular germ cell apoptosis and germ cell quality control in spermatogenesis [TP53 knockout can result in spontaneous testicular atrophy in rats [RELA, EGFR, MYC, AKT1, and so on, which might directly mediate the action of SJC on MI.Topological analysis was applied to the gene-pathway network to identify key targets of SJC in treating MI. ocesses. . RPS6 pae testis –57, and  control , implyinunctions , and MAPatogonia . In addiogenesis , and TP5 in rats . By inteRPS6, MAPK1, MAPK3, MDM2, DDX5, and TP53 might be the key target genes of SJC in the treatment of MI, and the drug-key compounds-hub targets-pathways network was constructed. However, these findings were not validated by in vivo and in vitro experiments, which need to be carried out in future studies.In our study, we investigated the potential pharmacology mechanism of SJC in treating MI using a network pharmacology-based approach. Firstly, a total of 314 targets affected by 97 bioactive compounds in the SJC were obtained. Quercetin, kaempferol, and luteolin regulated the most targets associated with MI. Secondly, 564 MI-related genes were collected and 413 candidate targets of SJC against MI were identified based on the analysis of the PPI network. Thirdly, GO and KEGG analysis suggested that SJC may treat male infertility through multiple biological processes including transcription, viral process, apoptotic process, and cell–cell adhesion and the related pathways including thyroid hormone signaling pathway and Hippo signaling pathway. Finally, pathway-gene network analysis indicated that Additional file 1: Additional file 2: Additional file 3: Additional file 4: Additional file 5: Additional file 6: Additional file 7:


Background: Coronary artery disease (CAD) is a common cardiovascular disease that has attracted attention worldwide due to its high morbidity and mortality. Recent studies have shown that abnormal microRNA (miRNA) expression is effective in CAD diagnoses and processes. However, the potential relationship between miRNAs and CAD remains unclear.Methods: Microarray datasets GSE105449 and GSE28858 were downloaded directly from the Gene Expression Omnibus (GEO) to identify miRNAs involved in CAD. Target gene prediction and enrichment analyses were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG).Results: There were nine differentially expressed miRNAs in CAD patients compared to the controls. A total of 352 genes were predicted and subjected to GO analysis, which showed that differentially expressed genes (DEGs) were mainly associated with axon guidance, neuron projection guidance, neuron-to-neuron synapses, and postsynaptic density. According to the KEGG pathway analysis, the most enriched pathways were those involved in transcriptional misregulation in cancer, growth hormone synthesis, secretion and action, endocrine resistance, axon guidance, and Cushing syndrome. Pathway analysis was mainly involved in the HIPPO and prion disease signaling pathways. Furthermore, a competing endogenous RNA (ceRNA) interaction network centered on miR-22-3p revealed eight related transcription factors in the cardiovascular system. The receiver operating characteristic (ROC) curve analysis suggested that miR-22-3p may be a better CAD predictor.Conclusion: The results indicate that miR-22-3p may function in pathophysiological CAD processes. Our study potentiates miR-22-3p as a specific biomarker for diagnosing CAD. Coronary artery disease (CAD) is a cardiovascular disease with a high global morbidity and mortality rate. Globally, it has caused serious social and economic burdens and has become a major health problem . AlthougSelective coronary angiography (CAG) is the current gold standard for diagnosing CAD. However, this surgical method is invasive, cumbersome, and expensive. It is mainly used in the late stages of the disease when multiple blood vessels are affected or arteries are seriously stenosed . At presIn recent years, deep sequencing and microarrays have effectively detected complex networks in the atherosclerosis process. They can be used as biomarkers in CAD patient diagnosis and prognosis . The comIn this study, we downloaded the original data of CAD and healthy control samples for microarray analysis from the GEO database. We used bioinformatics analysis methods to potentiate effective candidate biomarkers and key factors for early CAD screening and prognosis determination.http://r-project.org/) was used to download the coronary heart disease expression profile datasets GSE105449 , and 42 blood samples from healthy individuals not taking cardiovascular disease drugs (control 2). The GSE28858 dataset was a GPL8179-based validation set and included 12 blood samples from patients with cardiovascular disease and 12 blood samples from normal subjects  to analyze prediction efficacy. The AUC value is the total area covered by the ROC curve. A larger AUC value indicated a better classifier.We screened the GSE105449 dataset for DEGs by downloading the “limma” package , and heap < 0.05 was considered statistically significant. The DIANA TOOLS database showed pathway maps for pathways enriched in KEGGs  and visualized DEGs with transcription factors and the tissue association results using Cytoscape.We predicted the possible transcription factors of DEGs from the TransmiRv2.0 database  was used to analyze whether there was a statistical difference. Statistical significance between the two sets of categorical variables was compared and analyzed using a chi-square test or Fisher’s exact probability test. The correlation coefficients between different genes were determined using Pearson’s correlation analysis. All statistical p-values were two-sided, and statistical significance was set at p < 0.05.All data processing and analyses were performed using R software (version 4.0.2). For comparisons between two groups of continuous variables that conformed to a normal distribution, an independent Student’s The GSE28858 dataset was used as the validation set and included the case and control groups. Hsa-miR-22-3p expression in the samples of both groups was extracted, as the boxplot  and heatThe miRWalk website was used for DEG target gene prediction, and three DEGs  were included, with a validation level of 352 target genes predicted for enrichment analysis. GO analysis showed that the DEGs were mainly associated with axon guidance, neuron projection guidance, neuron-to-neuron synapses, and postsynaptic density . SupplemThe ceRNA interaction network of mRNA–miRNA-long intergenic non-coding RNA (lincRNA) was constructed using miR-22-3p as the center. As ROC curves of the GSE105449 and GSE28858 datasets were constructed with miR-22-3p as the center. The area under the AUC curve of GSE105449 was 0.719 , whereasNumerous studies have found that miRNAs play a large role in cell differentiation, biological development, and disease development. Evidence indicated that circulating miRNAs are crucial in CAD progression . HoweverIn this study, we identified potential miRNAs for the diagnosis and treatment of coronary heart disease based on two GEO datasets by analyzing differences in the expression of critical genes. However, the present study observed that PBMC miR-22-3p was increased in CAD patients compared to healthy control individuals (control 1 and control 2), suggesting that miR-22-3p upregulation might be crucial in the early stages of disease progression.Three sets of DEGs  were subjected to functional enrichment analysis. The aforementioned three miRNAs have been studied in the context of tumors, including breast cancer and intestinal tumors, and for their protective role in chemotherapeutic drugs against myocardial toxicity. Enrichment analysis was performed on 352 predicted target genes. The DEG-enriched pathways mainly included transcriptional misregulation, growth hormone synthesis, secretion and action, endocrine resistance, axon guidance, and Cushing syndrome. The Pathview-enriched pathways were mainly related to the Hippo signaling and prion disease pathways. The Hippo signaling pathway is a cellular signaling pathway that is significant in animal development and is responsible for regulating cell proliferation and organ growth . The corNFE2L2 gene, and the NRF2/ARE signaling pathway is considered a potential therapeutic strategy for antioxidative stress-mediated diseases, such as diabetes, fibrosis, and cancer (NFE2L2 polymorphism is associated with acute type A aortic coarctation risk and severity in a Chinese Han population (We constructed an mRNA–miRNA–lincRNA ceRNA interaction network centered on miR-22-3p and used the starBase v2.0 and miRWalk databases to predict eight related transcription factors associated with cardiovascular disease, namely, CTCF, JUN, JUND, NFATC1, NFE2L2, RAD21, RELA, and TAL1. Several studies have been conducted on the aforementioned hub genes in various diseases. For instance, CTCF is a key chromatin architecture protein that binds to insulators, regulates enhancer–promoter interactions for transcriptional insulation, and acts as a transcriptional repressor to regulate gene expression . More std cancer . NFE2L2 pulation . RAD21, pulation . HoweverIn conclusion, the present study suggests that miR-22-3p may be crucial in the onset and course of CAD. Our findings may provide potential targets for future CAD diagnoses and treatments.


Camellia oleifera (C.), which is extensively grown in south of China. The purpose of this study was to isolate new theasaponin derivatives from C. seed cake and explore their potential antitumor activity and their underlying molecular mechanism. In the present study, we first isolated and identified four theasaponin derivatives  from the total aglycone extract of the seed cake of Camellia oleifera by utilizing a combination of pre-acid-hydrolysis treatment and activity-guided isolation. Among them, compound 1 (C1) and compound 4 (C4) are newly discovered theasaponins that have not been reported before. The structures of these two new compounds were characterized based on comprehensive 1D and 2D NMR spectroscopy and high-resolution mass spectrometry, as well as data reported in the literature. Secondly, the cytotoxicity and antitumor property of the above four purified compounds were evaluated in selected typical tumor cell lines, Huh-7, HepG2, Hela, A549, and SGC7901, and the results showed that the ED50 value of C4 ranges from 1.5 to 11.3 µM, which is comparable to that of cisplatinum (CDDP) in these five cell lines, indicating that C4 has the most powerful antitumor activity among them. Finally, a preliminary mechanistic investigation was performed to uncover the molecular mechanism underlying the antitumor property of C4, and the results suggested that C4 may trigger apoptosis through the Bcl-2/Caspase-3 and JAK2/STAT3 pathways, and stimulate cell proliferation via the NF-κB/iNOS/COX-2 pathway. Moreover, it was surprising to find that C4 can inhibit the Nrf2/HO-1 pathway, which indicates that C4 has the potency to overcome the resistance to cancer drugs. Therefore, C1 and C4 are two newly identified theasaponin derivatives with antitumor activity from the seed cake of Camellia oleifera, and C4 is a promising antitumor candidate not only for its powerful antitumor activity, but also for its ability to function as an Nrf2 inhibitor to enhance the anticancer drug sensitivity.Theasaponin derivatives, which are reported to exert antitumor activity, have been widely reported to exist in edible plants, including in the seed cake of  Camellia is an economically and phylogenetically important member of the Theaceae family. There are more than 300 Camellia species widely distributed throughout China, Japan, and the tropical and subtropical regions of Asia + peak at m/z 571.4025 , corresponding to a molecular formula of C35H54O6. The IR spectrum indicated the presence of a hydroxyl group (3525 cm−1) and an α, β-unsaturated ester group (1710 and 1650 cm−1). The 1H NMR , an aromatic singlet at δH 5.31 , two isolated oxygenated methylene proton signals at δH 3.64  and 3.38 , three oxygenated methine protons at δH 3.75 , 5.80 , and 3.89 , six methyl proton signals at δH 1.05 , 0.98 , 0.93 , 1.40 , 0.85 , and 0.93 , as well as resonances for protons of an angeloyl group . The 13C NMR (δC 207.3 and 169.5), four olefinic carbons , four oxygenated carbons , eight methyls , eight methylenes , three methines  and six quaternary carbons , supported by the HSQC results. These data suggested that C1 contains an oleanane structure in the molecule, which is in accordance with the other reports  and two angeloyl substituent groups in C4. The glycosidic structure was connected to C-3 of the oleanane skeleton demonstrated by the HMBC correlations of H-a and C-3 as well as H-3 and C-a , 1.82 , and 1.87 , δH 169.6 (C-1′), 129.1 (C-2′), 139.2 (C-3′), 14.7 (C-4′), 12.5 (C-5′) for 2; δC 6.11 , 1.99 , and 1.89 , δC 169.0 (C-1′), 127.7 (C-2′), 139.2 (C-3′), 16.0 (C-4′), 20.8 (C-5′) for 3]. In compound 2, the HMBC correlation between H-16 and C-1′ was used to locate the angeloyl substituent group at C-16. In combination with the HMBC correlations, compound 2 was identified as camelliagenin A 16-tiglate. Likewise, the structure of compound 3 was determined as camelliagenin A 22-tiglate due to the HMBC correlations between H-22 and C-1′. Although 2 and 3 are known compounds that have been previously characterized elsewhere, their 1H and 13C NMR data were fully disclosed for the first time  using MTT assay. The widely used anticarcinogen cisplatinum (DDP) was used as the positive control. As shown in 1 could increase the cytotoxic activity in comparison with C2. Therefore, we hypothesized that the C-23 position could be a promising site of theasaponin scaffolds.According to the above results of the preliminary structure relationship analysis, we noticed that an additional aldehyde group at the C-23 position in C3 and C4 as potential promising antitumor compounds, we further investigated the effects of C3 and C4 on the expressions of proteins belonging to the signaling pathways related to survival, migration, and invasion in cells, such as the JAK2/STAT3, β-catenin-mTOR, Bcl-2/Bax-Caspase 3, and NF-κB/Nrf2 pathways.Considering the cytotoxic activities of the tested theasaponin derivatives and the importance of C3 and C4 , the value of Blank group was 9.32, and the values C4 (0.78125 μM) and C4 (1.5625 μM) were 10.7 and 9.73, respectively. This suggests that C4 can significantly promote early apoptosis. For late apoptotic cells , the value of Blank group was 9.63, and the value of C4 (0.78125 μM) was 12.5, which indicated that C4 (12.5 μM) could promote cell apoptosis. In addition, C4 promoted late cell apoptosis in a dose-dependent manner. To obtain more accurate molecular evidence of C4′s antitumor activity, typical apoptosis-related proteins were detected in HepG2 cells. The results in 4 promotes apoptosis by down-regulating β-catenin and up-regulating Caspase-3 and TNF-α.Cytometry A, the ap4′s antitumor activity, typical apoptosis-related proteins were detected in LPS-induced HepG2 cells. Nrf2 and its downstream protein HO-1 were tested. Inflammatory factors NF-κB, iNOS, and COX-2 were detected. As shown in 4 might be a suitable inhibitor of Nrf2, which could enable antitumor drugs to exert synergistic effects in cancer cells. The expressions of NF-κB, iNOS, and COX-2 were enhanced remarkably in the 6.250 group, and increased, though not significantly, in the 3.125 group , which were extracted from Camellia seed cake by the combination of pre-acid-hydrolysis treatment and activity-guided isolation. In addtion, molecular evidence shows that C4 has excellent antitumor ability, which can significantly inhibit inflammatory pathways and targets as well as regulating the intracellular redox balance.In the present study, four cytotoxic theasaponin derivatives were reported, including two new ones  in these five cell lines, indicating that C4 has the most powerful antitumor activity among them. The results indicated that C4 may trigger apoptosis through the Bcl-2/Caspase 3 and JAK2/STAT3 pathways, and that, more importantly, C4 can help overcome the resistance to cancer drugs by inhibiting the Nrf2/HO-1 pathway. Nrf2 is known as a master regulator of the redox balance. Nrf2 plays an important role in both tumor chemoprevention and tumor drug resistance, and some cancer cells can use this response to protect themselves from the damaging oxidative byproducts of abnormal metabolic activity and uncontrolled growth. Thus Nrf2 is traditionally considered to be a tumor regulator and an important indicator for the development of anticancer drugs. Our study indicates that C4 is a promising mediator for cancer chemoprevention.It has been reported that the main components of activity ,23. Like3 worked by down-regulating p-mTOR and p-STAT3, and C4 worked by down-regulating p-STAT3 and Bcl-2 in U251 cells. Compound 3 and Compound 4 both down-regulated p-mTOR, p-STAT3, and Bcl-2 in PAN02 cells. Cell injury activates the mTOR/STAT3 signaling pathway, thereby reducing cell apoptosis, which means that mTOR is an important signal transduction molecule. When mTOR is activated by external stimuli, the expression and activity of the downstream signaling molecules in the pathway will be increased, which can not only promote cell growth, proliferation, and differentiation, but can also regulate cell apoptosis. STAT3 can be activated by many cytokines and growth factors and is readily phosphorylated by the activated mTOR kinase. Activated STAT3 plays an important role in the control of cell growth, proliferation, differentiation, and apoptosis. Therefore, mTOR and STAT3 have crosstalk. In the future, we will conduct further studies to examine the effects of these components on the mTOR/STAT3 signaling pathway by using appropriate inhibitors, siRNA, or similar approaches.In the present study, Compound 4 can significantly stimulate the NF-kB inflammatory pathways and their targets and inhibit the Nrf2/HO-1 pathway to regulate intracellular redox balance, suggesting that it can be developed as a drug or functional food for more chronic diseases. Wang et al. have found that the glycosidic ligand on C3 of Camellia seed is the main source of its anticancer activity [3 and found that C3 also has antitumor properties and promotes cell apoptosis activity, but that it is not as effective as C4, which is consistent with Wang’s findings. This implies that C3 also has the potential to act as an anticancer drug or functional food. As a newly discovered compound, C1 has not been studied in its physiological function or effect for the time being. We plan to screen its active components and functions with the help of multi-omics technology.The development of cancer is also closely related to inflammation. For example, inflammation-related DNA damage in cancer stem cell-like cells leads to the development of cancers with aggressive clinical features . Some caactivity . During 4 can reduce the activity of Nrf2, and the effect was significant. This implies that C4 targeted the Nrf2 signaling pathway to promote tumor cell apoptosis and has the potential to develop as an Nrf2 inhibitor to anticancer. The present study supplied meaningful molecular data to promote the application of C4 in cancer drug development. Our study provided a new direction for this material, which is widely used in agriculture and in the food industry, as well as in new drug discovery.It is worth mentioning that, during the experiment, we found that C


Broilers provide a large amount of protein for human every year, in which skeletal muscle contribute a major role. In recent years, circRNAs have been found to be involved in skeletal muscle development, and more significant circRNAs in the process need to be revealed. In the study, transcriptome sequencing technology was used to obtain differentially expressed circRNAs (DECs) between fast- and slow-growing chickens. A variety of bioinformatics analysis were then used to identify the key circRNAs including novel_circ_0004547, novel_circ_0003578, novel_circ_0010289 and novel_circ_0003082. In addition, some candidate circRNA-miRNA pairs related to skeletal muscle were also obtained such as novel_circ_0002153-miR-12219-5p, novel_circ_0003578-miR-3064-3p and novel_circ_0010661-miR-12260-3p. This study would help to improve the regulation mechanism of circRNAs in skeletal muscle and also provide reference for broiler breeding.Skeletal muscle growth has always been the focus of the broiler industry, and circRNAs play a significant role in this process. We collected leg muscles of slow- and fast-growing Bian chicken embryos in the study at 14 (S14 and F14) and 20 (S20 and F20) days for RNA-seq. Finally, 123 and 121 differentially expressed circRNAs (DECs) were identified in S14 vs. F14 and S20 vs. F20, respectively. GO enrichment analysis for DECs obtained important biological process (BP) terms including nicotinate nucleotide biosynthetic process, nicotinate nucleotide salvage, and NAD salvage in S20 vs. F20 and protein mannosylation in S14 vs. F14. KEGG pathway analysis showed Wnt signaling pathway, Tight junction, Ubiquitin mediated proteolysis, and Notch signaling pathway were enriched in the top 20. Based on the GO and KEGG analysis results, we found some significant host genes and circRNAs such as NAPRT and novel_circ_0004547, DVL1 and novel_circ_0003578, JAK2 and novel_circ_0010289, DERA and novel_circ_0003082, etc. Further analysis found 19 co-differentially expressed circRNAs between the two comparison groups. We next constructed a circRNA-miRNA network for them, and some candidate circRNA-miRNA pairs related to skeletal muscle were obtained, such as novel_circ_0002153-miR-12219-5p, novel_circ_0003578-miR-3064-3p, and novel_circ_0010661-miR-12260-3p. These results would help to reveal the mechanism for circRNAs in skeletal muscle and also provide some guidance for the breeding of broilers. China is a major country for chicken consumption. With the improvement in people’s living standards, the demand for high-quality yellow feather broilers is becoming increasingly urgent. However, the growth rates of local chicken breeds in China are slow, and the feed utilization rate is low, so molecular breeding is needed to accelerate the genetic improvement of growth traits. Research on the genetic basis and regulation mechanism of growth traits of chickens will become an essential theoretical basis and premise of molecular breeding. The numbers of myofibers in birds are fixed late in embryonic development. Primary myofibers in chickens are formed around the embryonic age of 6 days, and secondary myofibers begin to differentiate at the embryonic age of 12–16 days, accounting for a majority of myofibers, which are basically fixed after the 20-day embryonic age [Circular RNAs (circRNAs) are biologically active nucleic acid molecules that are produced by precursor mRNA back-splicing in organisms ,4. The fIn recent years, circRNAs have been found to be involved in regulating many biological functions , includiThe slow- and fast-growing groups of Bian chicken were established by our team with myostatin gene-assisted selection . Then, tTotal RNAs were extracted from leg muscles. The sequencing cDNA libraries were then generated with the qualified RNAs in which ribosomal RNA (rRNA) was first removed . To seleRaw data (raw reads) of FASTQ format were processed, and clean data (clean reads) were obtained by removing reads containing adapter and ploy-N and removing low-quality reads from raw data. At the same time, Q20, Q30, and GC content of clean data were calculated. All of the downstream analyses were based on the clean data with high quality.p-value ≤ 0.05 were assigned as differentially expressed circRNAs (DECs) in the study. Finally, the gene ontology (GO) enrichment and KEGG pathway analysis of the host genes for DECs were implemented with GOseq (R-3.3.2) and KOBAS (v2.0), respectively.Paired-end clean reads were aligned to the reference genome GRCg6a using Bowtie 2 v2.2.8) . The cir . The cihttps://magic.novogene.com) (accessed on 13 September 2022). Finally, genes of GO_BP terms enriched in skeletal muscle development were selected for interaction network with Cytoscape (3.8.2).It is an important mechanism for circRNAs to function by regulating downstream target genes through recruiting miRNAs. MicroRNA target sites in exons of the 19 co-differentially expressed circRNAs were identified using miRanda software. GO enrichment analysis of miRNAs was then directly performed on the Novomagic platform  were selected for validation of RNA-seq in the two comparison groups . Specific divergent primers were designed with Primer 5.0 to confirm back-spliced junction (BSJ) sites of DECs. The PCR products were detected by agarose gel electrophoresis and sequenced with Sanger sequencing in Sango Biotech Co. Ltd. . RT-qPCR was performed on the Applied Biosystems 7500 platform  with reagent Taq Pro Universal SYBR qPCR Master Mix . Gene β-actin was used as the housekeeping gene, and relative expression was calculated with the 2t Test method. All data were presented as mean ± standard error (SE).Significant differences in body weight between slow- and fast-growing chickens were calculated using SPSS13.0 software with the Independent Samples p = 0.000) and S20 vs. F20 (p = 0.000). The bodyweights of slow- and fast-growing embryos at 14 and 20 days were as follows: S14: 9.26 ± 0.32 (g), F14: 11.25 ± 0.32 (g), S20: 32.60 ± 0.85 (g) and F20: 39.26 ± 1.20 (g). A significant difference in bodyweight was found between the slow- and fast-growing groups of 14-day embryos , as well as between 20-day embryos .Average egg weights before incubation were recorded as follows: S14: 46.07 ± 0.50 (g), F14: 54.50 ± 0.38 (g), S20: 45.89 ± 0.84 (g), F20: 56.16 ± 1.16 (g). Egg weights showed a significant difference in both comparison groups S14 vs. F14  were identified with 123 DECs  in compa123 DECs  in compa123 DECs . Compare123 DECs a. Simila123 DECs b. Cluste123 DECs  showed tp-value ≤ 0.05) in S14 vs. F14 and S20 vs. F20, respectively. Biological process (BP) terms in the top 30 of S14 vs. F14 contained formation of the primary germ layer, horizontal cell localization, positive regulation of transcription involved in the G1/S phase of the mitotic cell cycle, and regulation of cellular catabolic process  in comparison S20 vs. F20 were found, including nicotinate nucleotide biosynthetic process, nicotinate nucleotide salvage, pyridine nucleotide salvage, NAD salvage, and nicotinate nucleotide metabolic process. NAD serves both as a coenzyme for redox reactions, making it central to energy metabolism, and as an essential cofactor for non-redox NAD-dependent enzymes such as sirtuins. It can influence many cellular functions, including metabolic pathways, DNA repair, chromatin remodeling, cellular senescence, and immune cell function , and skeIn addition, some metabolic items are also greatly affected by NAD, including the primary metabolic process and cellular macromolecule metabolic process in S20 vs. F20 and regulation of the cellular catabolic process in S14 vs. F14. The genes enriched in them and their corresponding circRNAs may be very important for skeletal muscle, such as DVL1 and JAK2. DVL1, the host gene of novel_circ_0003578, is enriched in the above three items and has been found to inhibit human myoblasts . The hosAnother category of BP terms that accounts for a large proportion in the top 30 are “protein” related entries, including protein mannosylation in S14 vs. F14, and protein folding in the endoplasmic reticulum, protein maturation, chaperone-mediated protein folding, negative regulation of protein activation cascade, cellular protein metabolic process and protein folding in S20 vs. F20. Skeletal muscle is one of the most dynamic and plastic tissues. It comprises approximately 40% of total body weight and contains 50–75% of all body proteins . ERO1A wKEGG enrichment analysis showed that most of the pathways in the top 20 of the two comparison groups were related to skeletal muscle development, among which the same items included regulation of actin cytoskeleton, tight junction, ubiquitin-mediated proteolysis, and pentose phosphate pathway. Some KEGG pathways for host genes of DECs in the experiment were the same as those of other studies. Pan et al.  collecteProtein kinase C alpha (PRKCA), a subtype of protein kinase C (PKC), was enriched in 8 items in S14 vs. F14, including the mTOR signaling pathway, Wnt signaling pathway, and MAPK signaling pathway. PKCs are involved in multiple signal transduction systems that control cell proliferation, differentiation, survival, invasion, migration, and apoptosis . StudiesPPP3CB, the host gene of novel_circ_0008243, was enriched in Apoptosis, Wnt signaling pathway, MAPK signaling pathway, and Calcium signaling pathway in S14 vs. F14. It belongs to the protein phosphatase family, which could catalyze the dephosphorylation proteins. Protein phosphatases and kinases control multiple cellular events, including proliferation and differentiation, through regulating reversible protein phosphorylation, the most important post-translational modification . PPP1CB In comparison group S14 vs. F14, PTEN was enriched in the mTOR signaling pathway, Phosphatidylinositol signaling system, Tight junction and Focal adhesion. PTEN is a dual phosphatase with both protein and lipid phosphatase activities . It is aThree genes, DERA, ARHGEF7, and MGRN1, were enriched in the Pentose phosphate pathway, Regulation of actin cytoskeleton and Ubiquitin mediated proteolysis pathways in both of the comparison groups. Park et al.  identifiDVL1 and NAPRT have been discussed in the above BP item part. KEGG enrichment analysis showed that DVL1 was enriched in the Wnt signaling pathway of S14 vs. F14 and Notch signaling pathway of S20 vs. F20, while NAPRT was found in the Nicotinate and nicotinamide metabolism and Metabolic pathways in S20 vs. F20.CircRNAs can recruit miRNAs to regulate target gene expression , which iIn this experiment, we identified 123 and 121 DECs in comparison groups S14 vs. F14 and S20 vs. F20, respectively. Based on the important GO terms and KEGG pathways, we found some significant circRNAs. In addition, the circRNA-miRNA network of the 19 co-differentially expressed circRNAs revealed some skeletal muscle-related circRNA-miRNA pairs. These findings will guide our next research work and further help to reveal the development mechanism of circRNA in chicken skeletal muscle.


Heart failure (HF) is a globally prevalent cardiovascular disease, but effective drug targets and diagnostic models are still lacking. This study was designed to investigate effective drug targets and diagnostic models for HF in terms of miRNA targets, hoping to contribute to the understanding and treatment of HF. Using HF miRNA and gene expression profile data from the GEO database, we analyzed differentially expressed miRNAs/gene identification in HF using Limma and predicted miRNA targets by the online TargetScan database. Subsequently, gene set enrichment analysis and annotation were performed using WebGestaltR package. Protein-protein interactions were identified using the STRING database. The proximity of drugs to treat HF was also calculated and predicted for potential target therapeutic drug. In addition, further drug identification was performed by molecular docking. Finally, diagnostic models were constructed based on differential miRNAs. The GEO dataset was used to screen 66 differentially expressed miRNAs, incorporating 56 downregulated miRNAs and 10 upregulated miRNAs. The JAK-STAT signaling pathway, MAPK signaling pathway, p53 signaling pathway, Prolactin signaling pathway, and TGF-beta signaling pathway were enriched, as shown by KEGG enrichment analysis on the target genes. In addition, we found that 83 genes were upregulated and 92 genes were downregulated in HF patients vs. healthy individuals. Based on the inflammation-related score, hypoxia-related score, and energy metabolism-related score, we identified key miRNA-mRNA pairs and constructed an interaction network. Following that, TAP1, which had the highest expression and network connectivity in acute HF with crystal and molecular docking studies, was selected as a key candidate gene in the network. And the compound DB04847 was selected to produce a large number of favorable interactions with TAP1 protein. Finally, we constructed two diagnostic models based on the differential miRNAs hsa-miR-6785-5p and hsa-miR-4443. In conclusion, we identified TAP1, a key candidate gene in the diagnosis and treatment of HF, and determined that compound DB04847 is highly likely to be a potential inhibitor of TAP1. The TAP1 gene was also found to be regulated by hsa-miR-6785-5p and hsa-miR-4443, and a diagnostic model was constructed. This provides a new promising direction to improve the diagnosis, prognosis, and treatment outcome and guide more effective immunotherapy strategies of HF. Heart failure (HF) results from the dysfunction of diastolic and/or the systolic function of the heart; insufficient blood perfusion and blood stasis in the venous system in the arterial system would occur if the venous return blood could not be fully discharged from the heart, thereby causing cardiac circulatory disorder syndrome , 2. Inst2 [In a healthy heart, 90% of ATP production is produced via mitochondrial oxidative phosphorylation, and 60–70% of the energy was derived from lipid oxidation . Therefoβ signaling [At the posttranscriptional level, microRNAs (miRNAs) with approximately 22 nucleotides regulate expression of genes , 11. Sucignaling . TherefoThe heart, as a vital tissue that maintains blood circulation, ensures the metabolic needs. And it is too hard to obtain the tissue, especially in a healthy body. Based on the above statements, this study is aimed at mining key drug targets in HF using the NCBI Gene Expression Omnibus (GEO) dataset, including expression profiles of mRNAs and miRNAs. Differential miRNA and differential gene expression profiles of healthy controls and HF subjects were analyzed with the “Limma” package in R software. After that, we detected the key target gene of key miRNA, namely, TAP1. Finally, a diagnostic model was constructed based on the differential miRNAs. The current findings contributed to the development of understanding novel molecular mechanisms of HF pathogenesis, particularly the possible association of dysregulated pathways of the TAP1 gene with HF pathological processes, and ultimately predicted a new therapeutic target drug DB04847 for HF patients.Expression profile data were downloaded from the NCBI GEO database  for miRNHF with other diseases was excluded, and HF with survival information was retained.P < 0.05. We performed Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on miRNA target genes by the R software package clusterProfiler, filtered at P value < 0.05.Differentially expressed miRNAs were analyzed using Limma  and filtP < 0.05 and cor > 0.4 as the threshold. In addition, we also performed single-sample gene set enrichment analysis (ssGSEA) of KEGG-related pathways by the R language GSVA package and analyzed pathways that were statistically significant in HF patients and healthy individuals by t-test (P < 0.05).We downloaded energy metabolism-related pathways from the Gene Set Enrichment Analysis (GSEA)  website https://www.targetscan.org/vert_72/) regulatory database. TargetScan is a software for predicting miRNA binding sites in mammals. Before prediction of miRNA target genes, the 3′UTR region of the transcript needs to be determined first. The TargetScan database identifies the corresponding 3′UTR region of the transcript by a sequencing technique called 3P-seq  and provides a comprehensive sequence of the 3′UTR region by combining the analysis results of this technique with the available 3′UTR annotations in NCBI.The miRNA regulatory relationships were predicted from the online TargetScan  (https:/https://string-db.org/) database [https://string-db.org/) in this study, and based on the STRING database of protein interaction relationships, we filtered the interaction score≧400, that is, medium confidence.The STRING (database  supportsS (the gene set related to the treatment of heart failure), T (the set of drug target genes), D (the degree of the node of the related gene set in the PPI), and the distance d, which indicated the shortest path between node t and node s . The calculation was as shown below:ω is the weight of the target gene. The calculation method is ω = −ln (D + 1), otherwise if the target gene is a gene in the HF-related gene set, ω = 0.We calculated the proximity of drugs and its effect on treating HF. Here, we can give R). The distance d between these simulated drug targets (representing simulated drugs) and the TAP1-related gene set was then calculated, and after 10,000 random repetitions, a simulated reference distribution was calculated. Using both μd  and σd, the mean and standard deviation of the corresponding actual observation distance and the reference distribution were converted into a standardized score, that is, the degree of proximity z:We calculated simulated reference distance distribution corresponding to the drug. Protein nodes were randomly seen as the simulated drug target in the network, with the number of nodes keeping the same as the target scale  Homo sapiens, (2) resolution: 1.75 Å, (3) ligand: dual inhibitor of omapatrilat metalloproteinases ACE and NEP with Ki values of 0.64 nM and 0.45 nM, respectively, and (4) omapatrilat is a relatively mature NEP inhibitor, and the resolution of the crystal is 1.75, which is relatively low.P < 0.05 was considered as significant.ANOVA was conducted in comparing different groups containing multiple subgroups. First, we identified 66 differentially expressed miRNAs, of which 10 miRNA expressions were downregulated and 56 miRNA expressions were upregulated . Then, wP < 0.05). These genes were enriched to a total of two pathways . First, the differential analysis was performed for acute HF patients/chronic HF patients/HF patients vs. healthy individuals, respectively, and we found that 64 genes with downregulated expression and 36 genes with upregulated expression were obtained from the acute HF patients vs. the healthy group ; 17 genepathways , includipathways .To further understand the abnormal regulatory pathways in the organism triggered by HF, we performed ssGSEA of KEGG-related pathways and found that 23 pathway scores were significantly different between HF patients and healthy groups , includiHF leads to hypoxia , 27; theHF not only leads to hypoxia but also may affect the process of energy metabolism of the body. The corresponding scores of genes in these pathways SULFUR_METABOLISM, OXIDATIVE_PHOSPHORYLATION, and NITROGEN_METABOLISM were calculated by ssGSEA, the mean of which was taken as the score to indicate energy metabolism. We found a significant positive correlation between AGTR1 and energy metabolism and a significant negative correlation between EGR4 and PSD2 genes and energy metabolism by the correlation analysis.κB signaling, Jak/Stat signaling, B cell receptor signaling, and IL-6 receptor family. For the genes in the above related pathways, we calculated their related pathway scores by the ssGSEA method and found that a total of 24 genes were significantly associated with inflammation-related scores, hypoxia-related scores, and energy metabolism-related scores by correlation analysis . We concluded that the above TAP1-related gene sets were important genes for the treatment of HF and that drugs targeting these genes could have a greater impact on HF treatment development. Based on the drug target pairs in DrugBank and the predicted PPI interactions, we calculated the proximity of drugs and the effect on treating HF  that regulate TAP1 targets have a potential diagnostic value.


Mycobacterium tuberculosis) and host directed drug-target interaction network led to new candidate drug targets for drug repurposing studies. The current work sheds light on host genes and pathways enriched in active tuberculosis disease and suggest potential drug repurposing targets for host-directed therapies.Multi-drug resistant tuberculosis still remains a major public health crisis globally. With the emergence of newer active tuberculosis disease, the requirement of prolonged treatment time and adherence to therapy till its completion necessitates the search of newer therapeutics, targeting human host factors. The current work utilized statistical meta-analysis of human gene transcriptomes of active pulmonary tuberculosis disease obtained from six public datasets. The meta-analysis resulted in the identification of 2038 significantly differentially expressed genes (DEGs) in the active tuberculosis disease. The gene ontology (GO) analysis revealed that these genes were major contributors in immune responses. The pathway enrichment analyses identified from various human canonical pathways are related to other infectious diseases. In addition, the comparison of the DEGs with the tuberculosis genome wide association study (GWAS) datasets revealed the presence of few genetic variants in their proximity. The analysis of protein interaction networks (human and  Mycobacterium tuberculosis (Mtb) is the main causative agent of TB. Around 10% of individuals develop TB when exposed to Mtb and 5% of the infected individuals develop TB within 1-2 years while the remaining 5% develop the disease at any other time (Tuberculosis (TB) is an infectious disease which remained throughout human history. her time . Active her time . Individth leading cause of death globally in 2020. Around 86% of new cases reported around the world in 2020 were contributed majorly by China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh and South Africa with India leading the list  and also allow observations of such DEG upon vaccine/drug treatment. Further the DEG analysis also aids in understanding of regulatory mechanisms contributing to the functional consequences.In the current study, a statistical meta-analysis was carried out using whole blood expression profiles from infected TB patients to identify key human transcriptomics signatures characteristic of the disease. The study also utilized host genetic disease association and drug-repurposing analyses to further to prioritize the results. In addition, the gene ontology and pathway-based annotations identified genes and pathways significantly altered in the diseased condition.The whole-blood microarray gene expression profiles of patients with active pulmonary tuberculosis and healthy cohorts were retrieved from NCBI GEO . The datThe background corrected files were processed using limma package in R . The datStatistical meta-analysis was carried out using NetworkAnalyst . NetworkThe strength of the results obtained from meta-analysis was further validated by comparing the genes expressed in latent and control samples from the same datasets. Partial Least Square Discriminant Analysis (PLS-DA) was applied to the DEGs. Significant model was selected by 7-fold cross validation. The model performance was evaluated using the area under the Receiver Operating Characteristic (ROC) curve (AUC). All the above validation process was carried out using mixOmics package in R .Gene and pathway enrichment analysis was carried out using DAVID web server to identify significantly enriched Gene ontology (GO) biological processes (BPs) and KEGG pathways, which were ranked based on the hypergeometric test with FDR cutoff of 0.05 . DAVID wMtb protein interaction network (hmPPI).A comprehensive human protein-protein interaction network (hPPI) was constructed. High confidence, experimentally verified interactions extracted from STRING database was used for the construction . The STRThe highly interconnected components of the hPPI and hmPPI were identified using the Cytoscape plugin CytoHubba. CytoHubba is a user-friendly interface to explore important nodes in biological networks using various topological metrics. The hub genes of the networks were identified using the topological metrics degree and Maximal Clique Centrality (MCC) .The drug compounds interacting with DEG were retrieved from DrugBank Version 5 . DrugBanThe genetic differences between tuberculosis-affected and healthy individuals can give a mechanistic insight about the disease and functional implication of the affected gene. The single nucleotide polymorphisms (SNPs) proximal to the DEG were obtained from GRASP database  . GRASP dThe work plan and approaches implemented in the current study is illustrated in The database querying and filtering identified around 149 GEO microarray datasets for TB-related host response at the time of study (June 2022). Further, filtering based on the study inclusion criteria, a total of six datasets enclosing control and active TB samples were selected for next set analyses Table 1.The results of PCA indicated that the samples were clustered based on the observations of the study. However, after batch correction the samples were clustered based on the disease condition as active TB and control. We also observed a few samples outside the clusters before and after the batch correction procedure Figure 2When the datasets DEG were compared, we identified genes regulated in the same direction. The meta-analysis identified a total of 2038 DEG of which 861 genes were up-regulated and 1177 genes were down-regulated Table 2.via spliceosome, cytoplasmic translation and rRNA processing. Similarly for GO molecular function (MF) analyses, the DEGs were majorly enriched in RNA binding, protein binding and ATP binding. The GO CC analysis of down-regulated DEGs showed involvement in extracellular exosome, cytosol and lysosome. The GO BP analysis identified involvement in defense response to virus, innate immune response and response to virus. The GO MF analysis showed enriched functions such as protein binding, protease binding and MHC class I protein binding.The functional GO was carried out for the up and down regulated DEG identified by the meta-analysis Table 3.The pathway enrichment analysis implemented using DAVID identified various dysregulated pathways mediated by the DEGs Table 4.Salmonella infection, Hepatitis C and Epstein-Barr virus infection. We also observed the presence of Tuberculosis pathway in the list. The presence of tuberculosis pathway in our analysis indicated that the genes identified in the meta-analysis demonstrate their significant association with the disease.The down-regulated DEGs showed involvement in NOD-like receptor signaling pathway, Lysosome and other infectious disease pathways such as Influenza A, Identifying the physical interactions between the proteins will provide clues to combat infection. The mapping of 2038 DEGs along with their partners resulted in a network enclosing 325 nodes and 1460 edges Figure 5Mtb-host protein-protein interactions we did not observe any hub genes based on the topological metrics.The hmPPI interaction network enclosed 99 nodes and established 66 connections with an average of 1 connection between the neighbours Figure 6The DEGs from the study were queried against DrugBank to mine drugs targeting genes which may be used for repurposing against TB. A total of 22 drugs targeting 8 DEGs were obtained after the screening process Table 5.The detection of drug targets which has human genetic support by its involvement in the disease pathology may aid in success of the treatment against the disease by preventing late stage clinical failures. The evidence of involvement in the disease by the 2038 DEGs was retrieved from genome-wide association study (GWAS) datasets. A total of 483 TB-related SNPs were obtained from GRASP database. An overlap between the LD region with a SNP and the DEG location or the enhancer region suggest strong association between the SNP and that particular gene. A total of 33 genes showed association with TB-related SNPs Table 6.The meta-analysis of transcriptomes in this study identified S1PR1 as an up-regulated gene with highest fold change. S1PR1, by the detection of its ligand S1P in the blood and lymph, is crucial for naive lymphocytes to access the circulatory system. S1P-S1PR1 signaling is crucial for regulating immune cell development and function. S1P-S1PR1 signaling is needed for mature thymocytes to leave the thymus and for T/B cells to leave secondary lymphoid organs and enter the blood or lymph in both homeostatic and pathological situations . S1PR1 aMtb by controlling how Mtb moves through the lysosomes and stopping it from spreading in cells. Pre-mRNA splicing plays a crucial role in regulating gene expression and protein diversity. The Serine/Arginine rich (SR) proteins are the major components contributing to the selective splicing mechanism. The disruption in the RNA splicing mechanism can lead to crosstalk in the intricate network interactions. The Mtb infection alters the patterns of alternate splicing within the macrophages by affecting the expression of SR proteins -induced transmembrane (IFITM) gene family members. The IFITM members receive signals for their activation through type I and II IFN stimulation to preclude the establishment of productive infection. Ranjbar et al. show that IFITM proteins inhibit Mtb intracellular growth, indicating that they may contribute to host defense against intracellular bacterial infection  can be found in the article/Collected data, Implemented the analysis and Manuscript writing: NP. Conceived and designed the analysis: MA. All authors contributed to the article and approved the submitted version.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.We would like to thank Vellore Institute of Technology (VIT) for providing computational facility.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lymphedema is a chronic inflammatory disorder caused by ineffective fluid uptake by the lymphatic system, with effects mainly on the lower limbs. Lymphedema is either primary, when caused by genetic mutations, or secondary, when it follows injury, infection, or surgery. In this study, we aim to assess to what extent the current genetic tests detect genetic variants of lymphedema, and to identify the major molecular pathways that underlie this rather unknown disease. We recruited 147 individuals with a clinical diagnosis of primary lymphedema and used established genetic tests on their blood or saliva specimens. Only 11 of these were positive, while other probands were either negative (63) or inconclusive (73). The low efficacy of such tests calls for greater insight into the underlying mechanisms to increase accuracy. For this purpose, we built a molecular pathways diagram based on a literature analysis  of candidate and diagnostic genes. The PI3K/AKT and the RAS/MAPK pathways emerged as primary candidates responsible for lymphedema diagnosis, while the Rho/ROCK pathway appeared less critical. The results of this study suggest the most important pathways involved in the pathogenesis of lymphedema, and outline the most promising diagnostic and candidate genes to diagnose this disease. A chronic inflammatory disorder caused by abnormal accumulation of interstitial fluid resulting from ineffective flow and fluid uptake by the lymphatic system, lymphedema affects not only the lower limbs, but also the upper limbs, genitals, and face. By evolving in irreversible fibrosis, lymphatic damage, and tissue hypertrophy, it causes thickening and hardening of the limb ,2,3. LymThe aims of this study are twofold. First, we aim to assess to what extent the current genetic tests can detect causative mutations in primary lymphedema patients. Second, we aim to identify the candidate molecular pathways for lymphedema pathogenesis. At present only a few studies have focused on the pathways underlying this disease, which remain largely unknown. For these purposes, we used an up-to-date technique to detect genetic variants and an electronic search to build several molecular pathway diagrams, in order to define new diagnostic and therapeutic targets for lymphedema.n = 147) were recruited in clinics in the Italian regions of Campania, Latium, Lombardy, Marches, Puglia, Umbria, Veneto, and Trentino-Alto Adige. The recruited patients underwent several clinical investigations from 2019 to 2021 to diagnose primary lymphedema and exclude secondary causes of the pathology. The diagnosis of lymphedema was confirmed as reported in previous articles [Caucasian lymphedema patients n = 14 were recarticles . BrieflyTo meet the second aim of this study, we performed a search based on the diagnostic and candidate genes, as explained in the Materials and Methods section.We considered thirty-five genes to be correlated with several forms of primary lymphedema, following the criteria presented in Materials and Methods. The PI3K/AKT pathway ;RAS/MAPK pathway ;Rho/ROCK pathway ;Secondary pathways .We considered seventy-one genes as candidate genes, following the criteria presented in Materials and Methods. This study shows that the efficacy of genetic tests performed in a cohort of Italian lymphedema patients enables correct diagnosis in only 7% of the tested patients, which means that the majority of such patients do not have a specific and reliable genetic test for this disease. Despite past studies having attempted to relate genetic findings with key physiological or cellular mechanisms ,10,11, cThe most important pathway involved in lymphedema is probably the VEGF-C/VEGFR-3 pathway described in Apart from VEGF-C and VEGFR-3, many other proteins participate in this pathway, such as CCBE1, ADAMTS3, and PTPN14. CCBE1 is able to enhance in vivo sprouting of lymphatic vessels. A lack of it impairs VEGF-C and VEGFR-3 Erk signaling , while iHGF/MET signaling influences several biological activities, including differentiation, cell motility, growth, and survival . Moreove2+ cytosolic concentration. KIF11 heterozygous nonsense mutations [Activated by both VEGF-C/VEGFR-3 and HGF/MET signaling, the PI3K/AKT pathway is an essential component of the process of lymphangiogenesis, stimulating the survival of LECs  Figure . The relutations  cause miutations  cause lySeveral proteins encoded by the proposed candidate genes influences the activation of the PI3K/AKT pathway . In partThe RAS/MAPK pathway plays a pivotal role in promoting lymphangiogenesis . MutatioSeveral proteins encoded by the candidate genes participate in the RAS/MAPK pathway . Many reProteins encoded by the proposed candidate genes are involved in the Rho/ROCK pathway . AlthougMany transcription factors are involved in the function of the lymphatic system. Their mutations are linked to several diseases . In partApart from the diagnostic genes, several candidate genes code for transcription factors or influence their activity . For exaThe control of planar cell polarity (PCP) has a pivotal role in the function of the lymphatic system, with the relative mutations in genes involved in this pathway correlated with the development of primary lymphedema ,86. In pSeveral candidate genes are involved in the correct development of lymphatic valves. They can influence the PCP pathway (VANGL2 and SDC4), and the hippo pathway (DCHS1 and PPP1CB), or they can transduce mechanical tension in LECs (PECAM1 and CDH5) ,97,98,99Apart from lymphatic valves, lymph transport is enabled by active pumping, which stems from the rhythmic contraction of vascular smooth cells. The contraction of the lymphatic pumping unit, the lymphangion, enables the efficient pumping of lymph through lymphatic vessels up to the central veins. The malfunctions of lymph pumping affect in vivo and ex vivo lymph transport, causing lymphedema-related disorders ,103. DifSVEP1 and ACKR2 are involved in processes related to cell adhesion or macrophage recruitment to LECs. SVEP1 is a large extracellular mosaic protein with functions in protein interactions and adhesion, and its mutations have been found by a recent study in lymphedema patients . FurtherThe major limitation in this study is the lack of information for some diagnostic and candidate genes in the literature on their molecular activity. Thus, for this study, we relied on published papers that evaluate their cellular or tissue effects, and so, their physiological outcomes. Moreover, for many genes, molecular pathway data in LECs were unavailable; therefore, we relied on studies performed on different cell types. Finally, the provided list of genes should not be considered exhaustive, considering that further research could discover other possibly important genes and pathways involved in lymphedema.The first diagnosis of lymphedema is usually based on clinical symptoms, but many symptoms of this pathology are subclinical in its first stages. Thus, the molecular diagnosis of lymphedema could be useful in the scope of preventive medicine, and focusing our research on the molecular activity of diagnostic and candidate genes will permit the definition of new diagnostic and therapeutic targets.Ceasing to consider lymphedema only as a monogenic disease could be useful to explore new possibilities of diagnosis and treatment of this pathology still too much left aside. Lymphedema is now considered a Mendelian disorder, with variants of single genes being at the basis of its etiopathogenesis, but it could also arise from multigenic effects and from post-zygotic mutations. In multigenic diseases, polymorphisms of different genes, together with external factors, participate in the development of the pathology. In order to define the roles of specific genes in genetic disorders, the main strategy is to analyze their association with the disease in families with several affected individuals . PerformApproximately 5 mL of either peripheral blood or saliva  from proNegative when no variants were found in the analyzed genes;Inconclusive when the detected variants could be causative of the disease, but their effect is uncertain;Positive when the detected variants were correlated with the disease.We classified the genetic test as negative, inconclusive, or positive following the guidelines of the American College of Medical Genetics and Genomics , which sThe gene is involved in the development of the lymphatic system in vivo;Gene mutations cause in vivo lymphatic defects;The gene participates in a molecular pathway that is important in lymphedema pathogenesis.We searched for each of the identified genes in OMIM, Kegg, PubMed  and ScopThe low efficacy of established genetic tests for lymphedema calls for a deeper insight into the underlying molecular mechanisms, in order to define new diagnostic and therapeutic targets. In this study, we identified a few molecular paths that may explain the link between the observed genetic alterations and the physiological outcomes of lymphedema. The most important pathways involved in the pathogenesis of lymphedema are the PI3K/AKT and the RAS/MAPK pathways, while the Rho/ROCK pathway and other secondary pathways appeared less critical.


The aim of this study was to find underlying genes and their interaction mechanism crucial to the polycystic ovarian syndrome (PCOS) by analyzing differentially expressed genes (DEGs) between PCOS and non-PCOS subjects.P value and logFC. The screening threshold of DEGs was P < .05 and | FC | ≥ 1.2. GO annotation and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway enrichment analysis was performed by using DAVID (2021 Update). The protein-protein interaction (PPI) network of DEGs was constructed by using the STRING database, and the hub genes were recognized through Hubba plugin of Cytoscape software.Gene expression data of PCOS and non-PCOS subjects were collected from gene expression omnibus (GEO) database. GEO2R were used to calculating PCOS and non-PCOS subjects shared a total of 174 DGEs, including 14 upregulated and 160 downregulated genes. The GO biological processes enriched by DEGs mainly involved actin cytoskeleton organization, positive regulation of NF-κB signaling pathway, and positive regulation of canonical Wnt signaling pathway. The DEGs were significantly enriched in cytoplasm, nucleus and cytosol. Their molecular functions mainly focused on protein binding, calmodulin binding and glycerol-3-phosphate dehydrogenase activity. The PI3K/Akt signaling pathway and glycosaminoglycan biosynthesis were highlighted as critical pathways enriched by DEGs. 10 hub genes were screened from the constructed PPI network, of which EGF, FN1 and TLR4 were mainly enriched in the PI3K/Akt signaling pathway.In this study, a total of 174 DEGs and 10 hub genes were identified as new candidate targets for insulin resistance (IR) in PCOS individuals, which may provide a new direction for developing novel treatment strategies for PCOS. PCOS is an enigmatic endocrine disorder caused by hormonal imbalances and usually presents reproductive, metabolic and psychological syndromes. According to the Rotterdam ESHRE/ASRM Consensus, the diagnostic criteria of PCOS are hyperandrogenism, ovulation disorder, polycystic ovaries (detected by ultrasonography) and the exclusion of other endocrinopathies. The current understanding of the multisystemic features of this syndrome is increasing.,5 PCOS not only increases the risk of pregnancy-related disorders such as miscarriage, gestational diabetes, preterm birth and preeclampsia,–8 but also causes a range of other health problems, including chronic inflammation, insulin resistance (IR), glucose intolerance, metabolic syndrome and hypertension. Patients with PCOS often exhibit type 2 diabetes mellitus (T2DM), obesity and other metabolic disorders. PCOS is no longer simply considered an ovarian disease. Skeletal muscle IR has been reported in >60% of patients with PCOS and 10% of women with PCOS may develop T2DM by the age of 40 years. However, the molecule mechanism of IR in PCOS remains unclear and is worthy of further study. In this study, microarray data of PCOS and non-PCOS subjects were analyzed by using bioinformatics methods, which helped to explore critical differentially expressed genes (DEGs) underlying the pathogenesis of IR in PCOS.Polycystic ovary syndrome (PCOS), one of the most common endocrine disorders, is affecting up to 5% to 10% of women worldwide.We present the following article in accordance with the MDAR reporting checklist.https://www.ncbi.nlm.nih.gov/geo/) database by setting keywords as: “Syndrome, Polycystic Ovary” OR “Stein-Leventhal Syndrome” OR “Stein Leventhal Syndrome” OR “Syndrome, Stein-Leventhal” OR “Sclerocystic Ovarian Degeneration” OR “Ovarian Degeneration, Sclerocystic” OR “Sclerocystic Ovary Syndrome” OR “polycystic ovarian syndrome” OR “Ovarian Syndrome, Polycystic” OR “Polycystic Ovary Syndrome 1” OR “Sclerocystic Ovaries” OR “Ovary, Sclerocystic” OR “Sclerocystic Ovary” OR “PCOS” OR “polycystic ovarian syndrome”. GSE6798  and GSE8157  were downloaded for further analysis. Both datasets were based on GPL570 [HG-U133_PLus_2] Affymetrix Human Genome U133 Plus 2.0 Array chip. In addition, the PCOS patients meet the elevated free testosterone levels (>0.035 nmol/L) and hyperinsulinemia (>85 pmol/L); and the CON subjects meet regular menses, normal glucose tolerance, and no family history of diabetes. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).Microarray data of PCOS and non-PCOS subjects were obtained from the gene expression omnibus . The DEGs with P < .05 and FC ≥ 1.2 (logFC ≥ 0.3) were considered to be up-regulated genes, while the DEGs with P < .05 and FC ≤ 1.2 (logFC ≤ 0.3) were considered to be down-regulated genes.GEO2R was used to calculate the logFC (log2foldchange) and Common upregulated and downregulated genes between the two datasets were identified and defined as Co-differentially expressed genes (Co-DEGs). The Co-DEGs in the two datasets were displayed by Venn, and the volcano plots and heatmaps were drawn by using R-related visualization capabilities.Database for annotation, visualization, and integrated discovery (DAVID) (2021 Update) was used for gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. GO annotation is composed of biological processes (BP), cellular components (CC) and molecular functions (MF), and it is a good tool which can help to predict the main protein functions of DEGs. KEGG signaling pathway enrichment analysis is capable to classify all kinds of DEGs to specific pathways with system path. The DEGs were uploaded to DAVID (2021 Update) for GO annotation and KEGG signaling pathway analysis, then the top 10 GO annotation results were selected for bubble chart plotting.https://string-db.org/) database. The algorithm of the Hubba plugin of Cytoscape v3.9.1 was used for the top 10 hub genes analysis in the constructed PPI network.The PPI network of DEGs was constructed with the criterion of “confidence >0.7” based on the STRING  online tool. The GO annotation includes BP, CC, and MF, and the corresponding bubble chart was shown in Figure In total, 7 KEGG signaling pathways were enriched by DEGs, including PI3K-Akt signaling pathway, Focal adhesion, Gap junction, Propanoate metabolism, Glioma, Glycosaminoglycan biosynthesis and beta-Alanine metabolism. Corresponding detailed data were shown in Figure Based on the DEGs between PCOS and non-PCOS subjects, the PPI network of PCOS was constructed using the STRING database, which was displayed in Figure ,14 Currently, IR in PCOS patients is a hot topic in the field of endocrinology and IR appears to be the fundamental key factor within the pathophysiology of PCOS. Numerous studies have confirmed that high fasting glucose and increased serum insulin are commonly found in PCOS patients.,17 Impaired insulin responsiveness has been observed in skeletal muscle and myotubes of PCOS women. The skeletal muscle accounts for the largest portion of insulin-mediated whole-body glucose disposal, and thus, skeletal muscle IR is crucial for whole-body IR and PCOS. However, the research on the molecular level of IR in PCOS is lacking. In the present study, bioinformatics methods were used for analyzing the gene expression microarray data extracted from PCOS and non- PCOS skeletal muscle tissue. Ultimately, we detected a total of 174 co-DEGs by analyzing the gene expression microarray data from GSE6798 and GSE8157 for further study.PCOS is defined as a highly complicated endocrine disease in women, whereas the etiology and physiopathology have not been elucidated sufficiently.,21 Conversely, IR can also disrupt ovarian and uterine glucocorticoid receptor activation by regulating the NF-κB signaling pathway and then lead to PCOS. Zhao, Y et al demonstrated that the expression of WNT5a in PCOS patients was significantly elevated, and the up-regulated expression of WNT5a in PCOS increases inflammation and oxidative stress predominantly via the PI3K/Akt/NF-κB signaling pathway. In addition, previous studies have shown that the activation of WNT2/β-Catenin signaling pathway, was tightly associated with insulin resistance and estrogen deficiency, two hallmarks of PCOS. The Wnt signaling pathway is believed to be a significant contributor to the regulation of ovarian steroidogenesis, which could be one of the pathways modulated by gonadotropin signaling, and the cyclical changes of the endometrium are controlled by estrogen and progesterone via modulating the Wnt/β-catenin signaling pathway. Cytokine synthesis and increased endometrial inflammation in PCOS patients are coupled to the NFκB signaling pathway. The Wnt signal pathway might be involved in the apoptosis of granulosa cells and the progression of PCOS.,29The GO analysis results revealed that the genes with significant differences in expression were mainly involved in positive regulation of NF-κB signaling pathway, positive regulation of canonical Wnt signaling pathway and protein binding. It has been pointed out that the activated NF-κB signaling pathway might be one of the important factors in the pathogenesis of IR in PCOS.,31 the impaired PI3K/Akt signaling pathway has been implicated in the development of IR. In turn, IR would exacerbate the PI3K/Akt signaling pathway, forming a vicious cycle. Thus, the PI3K/Akt signaling pathway may act as a molecular link between PCOS and IR.Our KEGG enrichment analysis results suggested that many of EDGs were associated with the PI3K/Akt signaling pathway and glycosaminoglycan (GAG) biosynthesis, what is worth mentioning is that the PI3K/Akt signaling pathway was shown as the most enriched pathway. The PI3K/Akt signaling pathway is necessary for insulin stimulation of glucose transport,,35 The decrease of EGF and consequent inhibition of its downstream PI3K/Akt signaling pathway might be a feasible mechanism for IR induced by PCOS. The hub gene fibronectin (FN1), an integral component of mammalian extracellular matrices (ECM), is an essential glycoprotein that has numerous biological functions. FN1 not only regulates adhesion, motility, growth and development but also is related to metabolic syndrome (MetS) and IR.,39 In line with previous studies, the downregulation of FN1 seems to be related to PCOS and FN1 may regulate IR by affecting the PI3K/Akt signaling pathway. TLR4 is also a considerable hub gene, the downregulation of this gene can reduce the sensitivity of the insulin pathway by inhibiting the PI3K/AKT metabolic axis.,43 Glycosaminoglycans are long unbranched and complex polysaccharides that are also an essential component of ECM. It is by now generally accepted that GAG plays an important role in cell adhesion, migration, survival, and apoptosis. Research has shown that GAG is increased in skeletal muscle of insulin-resistant mice, indicating it may play a key role in the pathophysiology of PCOS.The PPI network of the hub genes further revealed that EGF, FN1, and TLR4 all participate in the PI3K/Akt signaling pathway, and they were shown to be significantly decreased in the skeletal muscle of PCOS patients. This is consistent with previous animal studies. Evidence has shown that mRNA expression of EGF was reduced in PCOS rats.Although the genes we explored might be promising targets for PCOS which can help to provide new directions for the pathogenesis and its molecule mechanism of PCOS, it is also essential to conduct deeper and more extensive research for more accurate relevant clinical treatment.In this study, a total of 174 DEGs and 10 hub genes were identified as new candidate targets for IR in PCOS individuals, and we indicated that the down-regulation of the PI3K/Akt signaling pathway may act as the underlying molecular basis of IR in PCOS, which may provide a new direction for developing novel treatment strategies for PCOS.Fei Zhou performed the data analysis and wrote this manuscript. Tiantian Cheng sorted out the data. Fei Zhou conceived and designed the experiments. Yuling Xing revised the manuscript. Linlin Yang and Huijuan Ma performed project coordination and supervised the project. All authors have seen and approved the final manuscript.Conceptualization: Fei Zhou.Data curation: Tiantian Cheng.Methodology: Linlin Yang.Supervision: Huijuan Ma.Writing – original draft: Fei Zhou.Writing – review & editing: Yuling Xing.


Aims: To evaluate the effectiveness and potential mechanism of calcium dobesilate (CaD) in diabetic kidney disease (DKD) patients.Methods: We searched for available randomized controlled studies on DKD patients’ treatment with CaD through open databases. Continuous variables were expressed as standardized mean difference (SMD) with a 95% confidence interval (CI). The putative targets and possible pathways of CaD on DKD were analyzed by network pharmacology. Molecular docking was employed to verify the match between CaD and the target genes.Results: In the meta-analysis, 42 trials were included, involving 3,671 DKD patients, of which 1,839 received CaD treatment in addition to conventional treatment, while 1,832 received conventional treatment. Compared with routine therapy, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) significantly decreased in the CaD treatment . The levels of serum cystatin C (Cys-C), urine levels of molecules reflecting kidney function  and micro glycoprotein), and inflammatory factors [hypersensitive c-reactive protein (hs-CRP)] were reduced compared with control groups, while glomerular filtration rate (GFR) was increased in patients treated with CaD for 12 weeks. CaD also showed a better effect on improving endothelial function. Network pharmacology results showed that the interaction pathway between CaD and DKD was mainly enriched in MAPK and chemokine signaling pathways. AKT1, CASP3, IGF1, MAPK8, and CCL5 might be the key targets for CaD in treating DKD.Conclusion: Combination with CaD is effective and safe in patients with DKD. Inhibition of MAPK and chemokine signaling pathways might be vital in treating CaD in DKD patients. Diabetes mellitus (DM) has afflicted around 422 million people worldwide and has become a leading cause of morbidity and mortality . DiabetiThe interstitial space grows in the first two phases, the mesangial volume rises, and the glomerular basement membrane thickens. They are silent stages because there is no detectable microalbuminuria in clinical practice, and there are currently no effective indicators for their detection . The earCalcium dobesilate (CaD) is a microcirculation-improving medication that improves hemodynamics, inhibits inflammatory responses, and suppresses interstitial fibrosis, among other things . CaD hasSome medications have a wide range of effects on humans, but they all point in the same therapeutic direction, implying that they can work on diverse targets in the same pathways. We have found that network-related approaches can be employed to emphasize our findings of drug action mechanisms in various data layers in the drug development process . Based oClinicaltrials.gov trials registry, and published review papers and editorials, was considered. The search terms were “Calcium Dobesilate” and “diabetic kidney disease” or “diabetic kidney disease” or “diabetic kidney disease” or “randomized” or “double-blind trial”, with no restrictions on subheadings or language. Other likely relevant citations were found in the reference lists of all included papers, and the literature not found in the abovementioned electronic databases was manually reviewed.The PRISMA  guidelines were followed in this meta-analysis . The PubThe inclusive criteria were as follows: 1) study subjects were diagnosed as DKD according to the corresponding guidelines, 2) all patients were randomized to receive treatment with CaD and contemporary medications or contemporary medications alone, 3) sample size in each study group was ≥ 15cases, 4) follow-up in each study group was ≥ 8 weeks; and 5) the outcomes were quantitative to facilitate outcome analysis.Exclusion criteria were as follows: 1) studies were nonrandomized or nonblinded, 2) patients enrolled had no definite diagnosis, 3) different medications were compared, 4) studies reported only symptomatic changes in patients without objective laboratory measurements; and 5) methodological quality was poor with a Jadad score <2.DKD is categorized into five stages according to the albuminuria-based classification. There was a hemodynamic alteration at the start of the first stage, with increased glomerular capillary hydrostatic pressure but no abnormalities in the ultrastructure. Hyperglycemic effects begin in the second part of the first stage, with thickening of the glomerular basement membrane, increased mesangial volume, and interstitial expansion. Because microalbuminuria cannot be measured in clinical practice and no suitable test marker has yet been discovered, the second stage is quiet . The earp ≤ 0.1, I2 ˃ 50%) were adopted for the random effects model. Statistical significance was defined as a two-tailed p < 0.05. The fail-safe number was used to estimate the extent of publication bias (RevMan version 5.3 was used to synthesize the data. Continuous variables were expressed as standardized mean difference (SMD) with a 95% confidence interval (CI). Chi-square and I2 tests were used to test heterogeneity. Nonheterogeneous results  were adopted for the fixed effects model, and heterogenous results  , Gene CaTo identify the interaction targets of CaD in the treatment of DKD, we selected the online drawing tool Interactive Venn  to draw p ≤ 0.01 as the cut-off threshold. Bioinformatics web toolsMetascapeThe integrated network of component–target–pathway was constructed using Cytoscape 3.7.1. The topology parameters of the network were analyzed with the built-in tool Network Analyzer in Cytoscape to identify the relationships of protein targets with components and the involved pathways.Suitable 3D structure “pdb” files of the core targets were downloaded from RCSB Protein Data Bank . PyMol2.A total of 939 trials were retrieved through database searches—PubMed (48), MEDLINE (17), EMBASE (25), Cochrane library (5), China National Knowledge Internet (248), VIP (219), Wanfang (277), and SinoMed (100). Five hundred and thirty-seven duplicate records were eliminated using endnote software. Subsequently, among the remaining 402 pieces, 123 trials were excluded from the primary screening according to the inclusion and exclusion criteria. Of the remaining 279 trials, 273 were evaluated as eligible, and 42 shots were finally included for meta-analysis ; Table 1p < 0.00001; I2 = 87%, BUN: SMD = −0.64; 95% CI, −0.96 to −0.32; p < 0.0001; I2 = 86%). The early phase was subdivided into 8-week and 12-week treatment cycles in most of the included literature. Furthermore, the results showed that CaD treatment for 8 weeks  and 12 weeks  could significantly reduce Scr and BUN levels compared to the control group. In the clinical stage of DKD, patients treated with CaD for 12 weeks also exhibited a statistically significant reduction in the expression level of the Scr and BUN compared with control groups . In the kidney failure stage of DKD, the data showed the same trend, that the groups treated with CaD for 12 weeks had a better effect on reducing Scr and BUN than comparators .Among all of the 42 trials, 38 trials evaluated the Scr and BUN. Thirty-five of the 38 trials were focused on DKD in its early stages. Two trials  focused p < 0.00001; I2 = 85%). Subgroup analysis was conducted to determine that the source of heterogeneity was from different treatment methods: one trial .Serum cystatin C (Cys-C) has been recognized as an ideal marker of kidney function . Nine trne trial  reportedne trial  focused ne trial  comparedp < 0.00001; I2 = 22%), while patients who received CaD for 8 weeks had increased GFR levels, but there was no statistical difference .Glomerular filtration rate (GFR) is widely recognized as a comprehensive measure of renal function, and its assessment is essential for clinical practice, research, and public health . GFR wasThe kidney function can be quantified by detecting the level of some molecules in urine. These molecules are divided into primary outcomes and secondary outcomes. The primary outcomes include urine albumin excretion rate (UAER) and 24 h urine protein/24 h urine albumin, and the secondary outcomes consist of alpha-1-microglycoprotein (α1-MG) and β2-microglobulin (β2-MG).p < 0.00001; I2 = 93%).Evaluated UAER, as the primary symptom of DKD, leading to a decrease of GFR and proteinuria, is regarded as the essential criterion for diagnosing DKD in the early stage . UAER wap < 0.00001; I2 = 94%)The data was reported in 12 trials , which ip < 0.0001; I2 = 91%). On the other hand, six trials .α1-MG and β2-MG are tubular markers and could be predictive in diagnosing DKD patients due to their accuracy . Four trx trials  reportedp < 0.00001; I2 = 0%) and decreasing ET  than the control groups.Endothelin (ET) and nitric oxide (NO) were used to show endothelial function. Three trials  reportedInflammation response was one of the mechanisms related to DKD development. By modifying vascular permeability; releasing vasodilator and vasoconstrictor mediators; causing kidney fibrosis; and inducing cytotoxicity, apoptosis, and necrosis in the pathogenesis and progression of DKD, it may have an impact on the glomerular filtration function . The enhp = 0.02; I2 = 96%).Four trials  were colp = 0.16; I2 = 99%).We collected two trials  involvinp = 0.35; I2 = 99%).Two studies  targetedp = 0.22; I2 = 98%).Blood viscosity can reflect the state of hemodynamics, and alternation of hemodynamics is one of the critical pathways in DKD pathological development. We found two studies  estimatiFor CaD, there are 293 related targets collected by searching the database. For DKD, we have retrieved 534 genes from the OMIM database, 1,678 genes from the Gene Cards database, and 1,189 genes from the DisGeNET database. After eliminating the redundancy, 2,771 known therapeutic targets were collected in this study. By drawing a Venn diagram to look for the intersection of CaD and DKD targets, 120 overlapping genes were obtained .To evaluate the role of potential targets in complicated diseases and discover connections, overlapping targets are submitted to the STRING11.0 platform to establish a PPI network. After analyzing the topology parameters of the PPI network, the 120 targets were sorted in descending order by degree and arranged in a concentric circle . Degree p-value.We conducted GO functional analysis and KEGG pathway enrichment analysis to elucidate the biological impacts of CaD on gene functions and signaling pathways of relevant targets in treating DKD. As shown in For biological processes, the targets were mainly enriched in the apoptotic signaling pathway, regulation of kinase activity, MAPK cascade, cellular response to chemical stress, and regulation of cytokine production. The top 10 items were mainly related to protein kinase activity, peptidase activity, and phosphotransferase activity  in molecular functions. The cellular components were primarily concentrated in cytoplasmic vesicle lumen, secretory granule lumen, ficolin-1-rich granule lumen, membrane raft, vacuolar lumen, lytic vacuole, and lysosome; according to the results of the KEGG pathway enrichment analysis, most were involved in the MAPK signaling pathway, PI3K-Akt signaling pathway, the Ras signaling pathway, focal adhesion, fluid shear stress, atherosclerosis, Rap1 signaling pathway, and the chemokine signaling pathway. Interestingly, the typical targets of CaD and DKD are mainly enriched in the MAPK signaling pathway, and protein kinase activity was mentioned in the top 10 molecular function items. Additionally, biological processes involved apoptosis signaling pathways, regulation of kinase activity, and MAPK cascade. Therefore, we surmised that the MAPK signaling pathway might be critical. The number of targets concentrated in the chemokine pathway was relatively large in the figure, and the biological process highlighted cellular response to chemical stress and regulation of cytokine production. Meanwhile, the meta-analysis results showed that CaD significantly reduced the level of inflammatory factors. These imply that the chemokine signaling pathway cannot be overlooked.A component–target–pathway network was constructed with Cytoscape3.7.1 based on the KEGG pathway enrichment analysis . Among tIn the present studies, the possible interaction activity between five key targets and their corresponding compounds of CaD was investigated with molecular docking verification. Among the docking results, most binding complexes possessed high binding affinity, averaging −5.12 kcal/mol. The modes of five binding complexes are displayed in in vivo, activating or inhibiting multiple metabolic and disease-related pathways, reminding us that a network pharmacological approach can be used to investigate the mechanisms by which drugs improve multiple metrics in patients.We gathered 42 trials of CaD intervention for DKD clinical treatment, including 3671 DKD patients in various phases of the disease, and divided them into the CaD experimental and control groups. The results showed that Scr, BUN, and Cys-C levels in the blood, as well as molecules including UAER, 24 h urine protein/24 h urine albumin, 1-MG, and 2-MG in the urine, were significantly lower in the CaD group compared to the control group, while GFR levels were substantially higher. These findings suggested that CaD could help DKD patients improve their glomerular filtration performance and kidney function. In addition, CaD could modulate endothelium contraction and relaxation by boosting NO and lowering ET, regulating microvasculature function. Inflammatory variables such as CRP levels also dropped in the CaD group, implying that CaD can help DKD patients lessen their inflammatory response. Compared to the control group, patients in the CaD group showed a trend of improvement in numerous indices, including glomerular filtering performance, endothelium function, and inflammatory function. As a result, we hypothesize that CaD may bind to multiple targets Then, using network pharmacology, we built a PPI network for the drug’s and disease’s shared targets and ran GO and KEGG analyses on these genes. According to BP enrichment, multiple genes are abundant in apoptosis, regulation of kinase activity, and cellular response to chemical stress, which could be CaD’s primary approach to suppressing the development of DKD. Based on the KEGG pathway enrichment analysis and literature research, we anticipated that CaD would play a therapeutic function in DKD primarily by modulating the MAPK signaling pathway and the chemokine signaling pathway . We idenReactive oxygen species (ROS), the products of normal metabolism and xenobiotic exposure, are significant factors related to the pathogenesis of DKD , and thePodocytes are terminally differentiated cells, and podocyte damage is the critical event leading to proteinuria in DKD . ROS is In addition tocell apoptosis, chemokine production has also been believed to play an essential role in the process of DKD . The proin vivo and in vitro (Although our results revealed the advantages of CaD for DKD, limitations did remain in the analysis. In the present study, we chose serum creatinine as a marker to assess renal function. At the same time, the literature has reported that CaD interferes to some extent with serum creatinine measured by sarcosine oxidase, both in vitro . Howeverin vitro . Other sin vitro . The evaIn conclusion, our research systematically elucidated the underlying molecular mechanisms by which CaD interfered with DKD based on the meta-analysis, network pharmacology, and molecular docking. We predicted five key targets from complex networks and concluded that CaD exerts a therapeutic effect on DKD by inhibiting MAPK and the chemokine signaling pathway. We expect this research to provide additional reference directions for CaD as a medicine in treating diabetic microangiopathy. Further exploration can be done in the future based on this study.