The study aimed to explore the key targets and molecular mechanisms of Dahuang-Tusizi drug pair (DTDP) in the treatment of diabetes nephropathy (DN) based on the GEO database by using network pharmacology combined with molecular docking and immune infiltration. The active components of the DTDP were screened using the Traditional Chinese Medicine Systems Pharmacology database and the Swiss Target Prediction database. The differential genes of DN were retrieved from GEO databases. Next, the intersecting targets of drug and disease were imported into the String database for protein–protein interactions network analysis, and the core targets were identified through topological analysis. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed with the help of the Metascape database and gene set enrichment analysis database. Subsequently, molecular docking was performed to verify the binding activity of the key component and the key target. The Nephroseq V5 database was used to verify the clinical relevance of DN and core genes. Finally, the Using CIBERSORT Algorithm to analyze the immune Infiltration of DN Gene Chip. The network analysis showed that 25 active ingredients of DTDP were associated with 22 targets in DN. The key active ingredients  and the core targets  were further identified. Enrichment analysis revealed signaling pathways including TNF, MAPK, and IL-17 signaling pathway. Molecular docking results showed that there was a strong affinity between the key components and core targets. The results of immune infiltration found that the proportion of macrophages in DN tissues was significantly increased. Our findings demonstrated that the characteristics of DTDP in treating DN are “multiple components, multiple targets and multiple pathways.” We predicted that DTDP may inhibit inflammation related pathways by regulating key genes, reducing macrophage infiltration. Thus, inhibiting inflammatory response to reduce glomerular damage and delay the development of DN. As the prevalence of diabetes has increased, the incidence of DN has increased rapidly. Being expected to 30% to 40% of diabetic patients develop DN, finally, the one third of DN patients develop end-stage renal disease. Currently, it has been shown that the mechanism of DN is the result of a combination of factors such as glucose metabolism disorders, changes in glomerular hemodynamics, inflammatory reactions, cytokines, endothelial damage, oxidative stress, ischemia and hypoxia, and genetic susceptibility. Chronic renal hypoxia, caused by various factors such as hyperglycemia, endothelial damage, and inflammatory responses, is one of the main causes of the occurrence and development of DN. Hypoxia is considered as an etiological factor in the progression of renal injury. HIF1α is a major transcription factor that transduces an array of cellular processes to let the cells adapt to hypoxic injury. Therefore, a sustained increase in HIF1α is a major adaptive stimulus to the hypoxic conditions. By regulating its target ZEB2 gene (Zinc finger E-box-binding homeobox 2), it affects pathological and physiological processes such as glucose, energy metabolism, inflammation, and plays an essential role in the occurrence and development of DN.–5 There is a growing body of evidence that hypoxia can cause kidney damage, inflammatory reactions, loss of podocytes and abnormal function, and reduced glomerular filtration rates leading to proteinuria. If not intervened in a timely manner at an early stage, this is a huge financial burden on society. Western medications are used to treat DN by regulating blood glucose, blood lipid levels and blood pressure. Early medical studies have shown that angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (angiotensin II receptor blockers) as first-line treatment for DN can reduce proteinuria, The role of delaying the progress of renal dysfunction. However, the side effects of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers limit the use of these drugs. Therefore, the search for safe and effective therapies for DN is of great urgency.Diabetic nephropathy (DN) is a common and serious microvascular complication of diabetes mellitus (DM). According to epidemiological studies, more than 425 million people worldwide are currently affected by diabetes. If no action is taken, more and more people with diabetes worldwide will rise to 629 million by 2045. Numerous experts pay considerable attention to exploring the Chinese medicine compound treatment of DN.–11 Dahuang-Tusizi drug pair (DTDP) consists of Dahuang and Tu-Si-Zi. As we all know, Dahuang (Rhubarb) is used as a cathartic in Chinese medicine. In modern pharmacology, Dahuang has long been used in China as an anti-bacterial, anti-inflammatory, anti-fibrotic and anti-cancer medicine.,14 It contains a variety of active ingredient, including anthraquinone, anthrone, acetophenone, flavonoids, and polysaccharides.–17 A research study indicated that Dahuang has a significantly inhibitory effect on the elevated metabolism of kidney, which effectively prevents the accumulation of extracellular matrix and promotes its effective degradation, modulates inflammation, delaying sclerosis of the kidney. Tu-Si-Zi (Semen Cuscutae) is a commonly used as tonifying the kidney in traditional Chinese herbal drug, prepared from the seeds of Cuscuta chinensis Lam,,20 which exhibits the anti-inflammatory, antioxidant, and anti-cancerous properties. Through clinical research, Tu-Si-Zi performed a bright future in the therapy of DM and its complications. In experimental study, it has been shown to improve kidney function, regulate the body’s endocrine and immune system. The chemical constituents of Tu-Si-Zi have been more comprehensive investigated, Its main components are flavonols such as quercetin, kaempferol, and sesamin, etc. These compounds may be responsible for the biological activity of the drug. The above research suggested that DTDP has a potential role in treating DN. Thus, the tonic of Tu-Si-Zi and laxatives of Dahuang were used together when treating renal disorders in traditional Chinese medicine.Nowadays, a number of evidence shows that traditional Chinese medicine (TCM) and herbal medicines have been playing an essential role in the treatment of DM and its complications. TCM is clinically proven effective and focuses on the functioning of the coordinated system.,27 It is employed to elucidate the interplay between the components, targets and pathways of the active ingredients of TCM, and it has been developed into a comprehensive tool to uncovered bioactive constituents and fundamental mechanisms of TCM from a holistic standpoint. Therefore, in this study, we investigated the assembly of “multi-component, multi-target” network of TCM compounds through network pharmacology and bioinformatics analysis. It aim to predict, reveal and clarify the molecular mechanism of DTDP in treating DN, hoping to provide more advice and guidance for the treatment of DN. The flowchart is shown in Figure Network pharmacology is an interactive drug-target-gene-disease based network that includes chemoinformatics, bioinformatics, network biology, and pharmacology.https://www.ncbi.nlm.nih.gov/geo/) using keywords such as “T2D diabetic nephropathy” and “glomeruli.” The probes are converted to the corresponding gene symbols based on the GPL17586 platform annotation information. With the help of the GEO2R software, the above chips are grouped, normalized and further processed. The screening conditions of differential genes were set as | log2FC |>1 and P < .05, and the significantly expressed differential genes (DEGs) were obtained. The differential gene heat map was drawn online by using bioinformatics platform (http://www.bioinformatics.com.cn/), and the original gene volcano map of the chip was drawn by using the mapping software GraphPad Prime 8.We retrieved the GEO database (https://tcmsp-e.com/). Assessing the efficacy of drugs administered orally into the systemic circulation is greatly reliant on oral bioavailability (OB), which stands as a crucial pharmacokinetic characteristic. Drug evaluates the potential of a compound to be a drug. Only molecules with increased OB and drug like (DL) can exhibit excellent pharmacological activity. We limited conditions of OB ≥ 30% and DL property ≥ 0.18; the 2D structures of active ingredients were retrieved through PubChem (https://pubchem.ncbi.nlm.nih.gov/), and their SDF files were downloaded. Then import the SDF file into Swiss Target Prediction (http://www.swistargetprediction.ch/) predicts potential drug targets. subsequently, the active ingredient and corresponding target of Cuscuta chinensis were imported into Cytoscape 3.8.0 software and the component target network was constructed.The active components of DTDP were downloaded from the Traditional Chinese Medicine Systems Pharmacology database  to construct the protein–protein interactions (PPI) network. The biological quality control is facilitated by the STRING database, which consolidates numerous networks of protein-protein associations. For the selection of the PPI target, we prioritize those with a confidence score exceeding 0.7 and other parameters remain default. The PPI network was screened by using Cytoscope and each node was scored using the plug-in CytoNCA. The Core networks were created and visualized using Cytoscape. The core targets in core network were scored according to the degree centrality (DC).The intersection of target genes were introduced into the STRING database (https://metascape.org/) along with Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. selected the filter condition organization as “hsa,” set Min Overlap = 3, P value Cutoff < .05, Min Enrichment = 1.5. The top 10 enrichment terms of biological process (BP), cellular component (CC), molecular function (MF), and KEGG enrichment analysis were presented in a bubble diagram using the bioinformatics platform. By utilizing the Cytoscape software, the KEGG relational network is established through the incorporation of either the Pathway ID number or the Pathway enriched genes. Next, an assessment of the network entails the computation of the neighboring nodes’ quantity, followed by the evaluation of the node size within the network, which is contingent upon the aforementioned neighboring node count.The intersection target of DEGs and DTDP was subjected to an analysis using the Metascape database  is a scientific technique used to analyze microarray data concerning genome-wide expression profiling. This method allows for the identification and sequencing of genes based on variations in their expression levels between 2 samples. In this study, GO and KEGG analyzes were performed on all detected genes using GSEA software (version 3.0). Unlike the GO and KEGG analyses, the GSEA enrichment analysis does not necessitate a differential gene threshold. It effectively assesses if a group of genes is distributed randomly within a specific phenotype or is subject to abnormal regulation, thereby ascertaining the biological significance. Consequently, we utilized GSEA to explore the variances in biology between samples of DN and regular kidney tissues.https://www.rcsb.org/) database to obtain the core target protein PDB format. Pymol software was used to remove water molecules and short-molecule ligands of the proteins. The docking process combined the AutoDock tool with the currently widely used molecular docking strategy-AutoDock Vina, and improved the speed and accuracy of molecular docking through various evaluation functions. The edited results were done by Vina. Binding of drug to targets can be visualized using heatmaps. Using the Pymol molecular alignment visualization software, our findings indicate that the lower the binding energy, the better the alignment.To obtain the 2D structure of the key components of DTDP, we imported the key active ingredients into PubChem and converted the 2D structure to 3D using chem3D software. Meanwhile, we used the PDB (http://v5.nephroseq.org) is an extensive noncommercial platform for exploring and analyzing gene expression data related to kidney disease. To ensure the reliability of our findings, we assessed the expression levels of essential genes linked to DN in comparison to those of normal controls, employing Nephroseq V5 database. We considered a P value lower than .05 as indicative of statistical significance.The Nephroseq V5 online database (http://humphreyslab.com/SingleCell/). The core genes were first searched by entering each of the 8 core genes in the home page of the Kidney Interactive Tranomics database to analyze the expression of the core genes in a cluster of single kidney cells. The output results in 3 images, which are the single-cell clustered t-SNE map of the dataset, the t-SNE map of the core genes, and the core gene expression violin map for each cell class group.Online database for analysis of renal single-cell datasets: the Kidney Interactive Tranomics database (http://www.bioinformatics.com.cn/keywords=ROC), and the diagnostic value of the core genes was assessed through the AUC values.ROC (receiver operating characteristic) analysis for diagnostic biomarkers is a common method for evaluating diagnostic accuracy, characterized by the combination of sensitivity and specificity. In medical research, ROC curves have been used to assess the diagnostic efficacy of core genes, to study the predictive accuracy of core genes for diseases, and to determine cutoff values. The corresponding sensitivity and specificity are calculated at different boundary points in a continuous variable, with sensitivity in the vertical coordinate and 1 − specificity in the horizontal coordinate, as indicated by the larger area and better sensitivity under the ROC curve. Plotting the ROC curves of core genes versus DN was carried out through the microbiology letter . the results showed the total of 32,658 genes were obtained by microarray analysis of the GSE96804 gene chip, and the DEGs screening conditions were set to |log2FC|>1 and We obtained the active ingredients of DTDP with OB ≥ 30% and DL ≥ 0.18 as the screening cutoff point. A total of 25 active ingredients were obtained: 15 active ingredients from Dahuang and 11 active ingredients from Tusizi; while MOL000358 was consensus components. These active phytochemicals are given in Table A Venn diagram was constructed by intersecting the up-regulated and down-regulated genes in the DEGs with the targets of DTDP. Analysis of Figure To conduct a thorough investigation of the 22 crossover genes, we utilized the String online database for PPI analysis Fig. A. The inWe screened the key drug component networks corresponding to the core targets from the above networks. In addition, a hub compound-target network was constructed between 19 active phytochemicals and 22 potential anti-DN core targets, as shown in Figure A total of 35 signaling pathways were associated with the effectiveness of DTDP in treating DN. Bubble plots of the top 10 entries were shown in Figure Compared with the control group, Figure A large rectangular area was occupied by genes including JUN, FOS, CCL2, PTGS2, MMP2, CXCR1, CXCR2, EGF, KDR, CD44, and CYP2B6, as depicted in Figure -1) with 6 key active components. The results are shown in Figure −1.Using molecular docking analysis, we conducted an docking on the significance of 6 vital components in the DTDP and the interplay with 8 essential targets found in DN. By utilizing the PDB database, we identified protein crystals that corresponded with the central target genes. The PDB with the crystal structures of JUN, FOS, MMP2, CCL2, CD44, KDR, PTGS2, EGF are 1jnm, 1s9k, 1qib, 4zk9, 2i83, 6xvk, 5f19, 2kv4. All of the above major targets showed strong binding affinities . The above results showed the hub genes plays an important role in DN glomerulopathy.The results showed that hub genes  were significantly expressed between DN and normal donors Fig. K. We fouSingle cell cluster analysis can understand the expression of genes in each cell. According to the results, EGF is significantly expressed in the LOH loop and distal convoluted tubules of DN, KDR is expressed in ENDO, and JUN and FOS are widely expressed in all cells of DN. The results are shown in Figure The area under the ROC curve, known as AUC, is currently considered a standard method for evaluating the accuracy of predictive distribution models. The curve areas of FOS (AUC = 1), JUN (AUC = 0.978), PTGS2 (AUC = 0.927), CD44 (AUC = 0.727), CCL2 AUC = 0.768), MMP2 (AUC = 0.904), EGF (AUC = 0.884), and KDR (AUC = 0.818) are all above 0.8, further proving the reliability of the above genes as core target genes. The results are shown in Figure 8, MMP2 (P < .05) in the abundance of 8 immune cells. Specifically, the percentages of M2 macrophages, CD8 T cells, neutrophils, activated mast cells, and resting mast cells were found to be substantially higher in DN tissue samples compared to the control group. These findings implied that macrophages and other immune factors may have a particularly consequential role in the onset and progression of DN.The CIBERSORT algorithm was employed to analyze the GEO data, and boxplots illustrating the proportion of 22 distinct immune cells were generated using the R language. Figure P < .01), among which CD4, MMP2, CCL2 with M2 macrophages had a strong positive correlation (Corr > 0.8), EGF and KDR showed a strong negative correlation with M2 macrophages (Corr < -0.7). FOS, JUN, PTGS2 were strongly positively correlated with neutrophils (Corr > 0.8), CD4, MMP2, CCL2 was negatively correlated with neutrophils (Corr < -0.6).The Figure  oxidative stress, accumulation of advanced glycation end-products, activation of protein kinase C, overexpression of transforming growth factor-β, followed by increase of extracellular matrices. In the past few years, substantial evidence has substantiated the notion that anomalies in immune infiltration and inflammatory reaction could play a pivotal role in DN. The occurrence of DN inflammation might involve various types of cells, including renal endothelial cells , as well as exogenous cellular elements . These diverse cell types actively contribute to the progression of DN. Furthermore, some pro-inflammatory molecules such as adhesion molecules, chemokines and cytokines play an important role in the development of DN. Therefore, these proinflammatory molecules and inflammatory cells may be current therapeutic targets for DN.DN is a serious microvascular complication in DM patients. Several factors are involved in the development of DN, including genetic factors, glomerular hyperfiltration,Network pharmacology methods and bioinformatics analysis help to identify putative active ingredients and drug targets. First, in this study, Using GEO2R software to analyze the GSE96804 chip, a total of 654 genes were obtained. There are 314 up-regulated genes and 340 down-regulated genes in DEGs. Subsequently, the Traditional Chinese Medicine Systems Pharmacology database was searched, and a total of 25 active DTDP components were found. At same time, a total of 431 DTDP candidate targets were selected through Swiss Target Prediction. After interleaving the DEGs with the drug target genes, 22 genes were involved in DTDP treatment of DN, with 4 showing upregulation and 18 displaying downregulation, we further explored the PPI core genes and constructed a core gene network. After DC screening, the core genes greatly related to the treatment of DN were finally obtained, including JUN, EGF, CD44, FOS, KDR, CCL2, PTGS2, MMP2 and additional genes formed the core of the network. Among them, CD44, CCL2, PTGS2, MMP2, and JUN were mainly related to the inflammatory response. It is largely involved in the transcriptional regulation of multiple genes such as cell survival, proliferation and apoptosis. The activation of JUN is critical for mesangial cell proliferation and extracellular matrix production, and mesangial expansion is an important pathological feature of glomerulonephropathy. CCL2, also known as MCP-1, represents a potent type of chemokines, primarily synthesized by tubular epithelial cells and mesangial cells. It is also thought to be an inflammatory factor that promotes DN and is elevated in DN patients.,40 CD44 is a type I transmembrane glycoprotein family known as an adhesion molecule. It has a broad distribution in various tissues, including leukocytes, epithelial cells, and endothelial cells. Additionally, CD44 plays a significant role in multiple physiological and pathological processes. These include interactions between cells and between cells and the extracellular matrix, the extravasation of leukocytes, wound healing, cell migration, and the activation of lymphocytes. A study reported that CD44 expression is markedly enhanced in glomeruli and on injured renal tubular epithelial cells and capillary endothelial cells. PTGS2, also known as prostaglandin biosynthesis enzyme, plays a pivotal role in the production of prostaglandins. These prostaglandins have critical implications in processes like inflammation and cell division. Moreover, PTGS2 exhibits various biological actions, including the suppression of apoptosis, facilitation of cell growth, and stimulation of blood vessel formation. These findings imply a significant involvement of PTGS2 in the formation and progression of DN. The EGF is a potent trophic factor produced mainly in kidney tubules, and plays a role in kidney development and in tissue repair. Experimental studies have shown the importance of EGF and KDR signaling in maintaining tubular epithelial cell integrity. In addition, it was demonstrated that matrix metalloproteinases (MMP) levels are implicated in diabetic complications such as retinopathy, cardiomyopathy, peripheral neuropathy and nephropathy. The evidence suggested that MMP-2 is linked to renal hypertrophy and abnormal extra cellular matrix deposition, which are hallmarks of DN. To confirm the robustness of our results compared to external datasets, To examine the expression levels of core genes linked to DN and normal controls, we employed the Nephroseq V5 online database. Statistical significance was defined as a P value less than .05. In our study, it was shown that between DN and normal living donors, core genes were significantly expressed. We found that 6 core genes  were significantly larger than normal renal samples. Two hub genes (KDR and EGF) were significantly lower than that of normal kidney samples and are statistically significant (P < .05). Both the literature reports and the V5 results showed that the expression of JUN, FOS, MMP2, PTGS2, and CD44 were up-regulated in DN. However, KDR and EGF were downregulated in DN.According to a research study, it has been demonstrated that JUN (AP-1 transcription factor) represents a heterodimer formed through the combination of FOS protein and JUN protein encoded by the proto-oncogene. The administration of Quercetin significantly enhances disease symptoms, renal hypertrophy index, renal histopathology, oxidative stress, and blood glucose levels in a mouse model of DN. Sesamin, a natural lignin compound, and it possesses health beneficial effects, such as anti-aging, anti-cancer, anti-diabetes, anti-inflammatory and antioxidant properties. The incorporation of Sesamin as a natural adjuvant can be effective in improving vascular reactivity and aortic permeability and DN. Eupatin is a polymethoxylated flavonoid that is said to inhibit iNOS and AchE. The studies have shown that eupatin can significantly inhibit the expression of iNOS and COX-2 in macrophages and microglia, as well as the production of nitrite induced by LPS, and has a significant anti-inflammatory effect. In summary, we speculated that the above key ingredients treatment of DN through anti-inflammatory effects.Simultaneously, we screened 6 key active components from the core of the gene network, such as sesamin, quercetin, EUPATIN, SennosideE_qt, SennosideD_qt and palmidin A. Among them, sesamin, quercetin and EUPATIN have been widely reported in experimental studies for their anti-inflammatory effects. Many studies have shown that Quercetin is a natural flavonoid compound, which has various pharmacological effects such as anti-oxidation, anti-inflammation, anti-cancer, anti-diabetes, and anti-obesity. Hence, we concluded that DTDP treatment of DN by inhibiting inflammation pathways, reducing extracellular matrix (ECM) deposition. From the enrichment analysis of KEGG, the therapeutic targets of DTDP against DN were mainly enrichment in inflammatory pathways, including MAPK signaling pathway, TNF signaling pathway, IL-17 signaling pathway, advanced glycation end-products-RAGE signaling pathway in diabetic complications, etc. Based on the enrichment analysis of GSEA, the DN samples were mainly associated with the MAPK signaling pathway. Thus, we speculated that DTDP treatment of DN were mainly enrichment in MAPK signaling pathway. As we all know, the MAPK signaling pathway is canonical inflammatory signaling pathway. Many studies have supported that MAPK signaling pathway plays a key role in inhibiting apoptosis, promoting proliferation and inhibiting inflammation in DN rats.,56 Therefore, inhibition of the MAPK signaling pathway can attenuate the inflammatory response of DN and protect the kidney.Meanwhile, In this study, DTDP treatment of DN was subjected to both GO enrichment analysis and KEGG enrichment analysis. The results of GO-BP enrichment analysis revealed that the treatment with DTDP for DN primarily targets inflammatory responses, vascular development, leukocyte migration, breakdown of the extracellular matrix, regulation of cell-cell adhesion, and migration of endothelial cells. Combined with the enrichment analysis of the GSEA, both the DEGs and the GSEA of the GO-BP analysis focus on inflammatory responses. Thus, DTDP plays a role in treating DN through inhibiting inflammatory response. In addition, The GO-MF mainly focuses on serine-type endopeptidase activity, positive and negative regulation of RNA polymerase II-specific DNA-binding transcription factor binding, cytokine receptor activity, oxidoreductase activity, etc. The GO-CC were mainly located in extracellular matrix, secretory granule membrane, and external side of plasma membrane, etc. According to DEGs and GSEA-GO enrichment results, inflammation is closely related to the occurrence and development of DN and it is characterized by excessive deposition of ECM proteins in the mesangium and basement membrane of the glomerulus and in the renal tubulointerstium. During the pathogenesis of DN, inflammatory cells proliferate and differentiate, renal tissue cell infiltration increases, and the large amounts of cytokines are secreted to mediate renal tissue injury. It has been reported that macrophage infiltration, one of the hallmarks of DN, is significantly increased in the glomerular tissues of most people with DN. As an essential component of the mononuclear phagocytic system, macrophages play a highly critical role in the development of the body, tissue homeostasis and repair. In general, macrophages have 2 phenotypes, M1 macrophages and M2 macrophages, with distinct functions. M1 macrophages are typical activated cells that secrete so much pro-inflammatory cytokines , Chemokine (MCP-1) and reactive oxygen species promote inflammatory responses that cause additional damage during the pathogenesis of DN. M2 macrophages frequently function as immunosuppressive cells involved in anti-inflammatory responses, tissue remodeling, and tumor progression. Therefore, our focus was on the significant increase of M1 macrophages observed in glomerular samples obtained from patients with DN. Finally, Studies have shown that the docking fraction of the bioactive components to the target structure is less than -7kcal mol−1, indicating that their binding affinity is stronger, that is, the smaller the docking fraction, the stronger the binding strength and the lower the binding force. In our study, we screened 6 active ingredients  and 8 core targets  were verified by molecular docking technology. The result shows that the binding energies of docking are less than −6 kcal·mol−1, which means the key active components of DTDP have excellent binding activity with key targets of DN. Among them, Sesamin had the best binding effect on the 7 core target proteins, and the docking scores were all below −8.0 kcal mol−1, indicating a significant impact of DTDP treatment on DN. The docking results offer an effective approach to predict the binding mode between DTDP and core DN-associated target proteins, providing valuable guidance for further experimental validation.In this study, we used the CIBERSORT algorithm to analyze the immune cell infiltration in the above-gene chip . The results showed that the proportion of M1 macrophages in the DN tissue sample was significantly higher than in the control sample. The macrophages were observed in the glomeruli and interstitium of DN patients, suggesting that immune factors such as macrophages may play a critical role in the development of DN.However, due to time and funding issues, we only explored a network pharmacological analysis of the potential functional mechanism of action of DTDP on DN. This study has not been verified by experimental data, and our study still has certain limitations. First, additional invitro experiments and clinical validation are needed to support our future research. Second, DEGs were identified by analyzing microarray data published in the GEO database. However, the sample size of DEGs related to DN is small and needs to be strengthened in the future. And, as injury targets for DN, renal innate cells are involved in disease onset and development. Nonetheless, the study did not address how kidney cells and macrophages communicate. Therefore, we will continue to explore the relationship between DTDP and DN in the following future studies. We will investigate the therapeutic effects of DTDP on DN in in vitro experiments and clinical validation. We will investigate the involvement of intrinsic cells of kidney and macrophage cultures in the inflammatory response mechanism. We will compare the epigenetics related database of DN with the active components of DTDP to explore the potential mechanism of DTDP on DNA methylation, histone modification, and noncoding RNA in DN. The molecular mechanism of DTDP in the treatment of DN has been further investigated by means of gene silencing, CO-IP, EMSA, etc.In summary, this study combined network pharmacology and bioinformatics to explore the mechanism and molecular targets of DTDP against DN. Our results revealed that DTDP exerts pharmacological effects on DN are “multiple components, multiple targets and multiple pathways.” At the same time, we predict that DTDP may inhibit the inflammatory response by modulating MAPK and TNF inflammatory signaling pathways, reducing macrophage infiltration, thereby reducing glomerular damage and delaying the onset of DN. Thus, DTDP and its key components may be promising drugs for the treatment of DN. And the mechanism of action is shown in Figure Conceptualization: Wenjing Liu.Data curation: Wenjing Liu.Funding acquisition: Ling Yuan.Project administration: Ling Yuan.Software: Shaozhang Hou, Mengying Che.Visualization: Fandi Men, Duojie Xu.Writing – original draft: Wenjing Liu.Writing – review & editing: Ling Yuan, Yi Nan.


Lymphoma of the conjunctiva is an ocular malignancy derived from clonal proliferation of lymphocytes. The majority of conjunctival lymphoma is extranodal marginal zone B-Cell lymphoma (EMZL), however diffuse large B-cell (DLBCL), follicular (FL), mantle cell (MCL) and T- cell subtypes are also seen. Clinical manifestations are non-specific, but include unilateral or bilateral painless salmon-pink conjunctival lesions. Approaches to treatment have centered around local immunomodulation, often with Interferon-α2b or Rituximab  with or without radiation. Although conjunctival lymphoma is generally considered an indolent disease, recent advances in next-generation sequencing have improved clinicians’ ability to predict future recurrence or systemic disease through assessment of cytogenic and molecular features. In this paper, we review the classification, clinical features, diagnostic techniques, and emerging strategies for management and prognostication of conjunctival lymphomas. Lymphomas are malignant lymphoid tumours arising as clonal proliferations of either B-lymphocytes or T-lymphocytes, or less commonly natural killer (NK) cells. When occurring in the conjunctiva, they can be primary well-defined tumours, or secondary manifestations of systemic lymphoma. Although lymphoma in this region accounts for only 5–10% of all extranodal lymphomas, it is one of the most common adnexal malignancies . On clinThe conjunctiva contains specialised lymphoid tissue, and acts as an antigen barrier as part of the mucosa-associated lymphoid tissue (MALT) system . AntigenMorphologically, the lymphoma cells infiltrate around reactive B-cell follicles and spread to form confluent areas of heterogeneous small B-cells  with scattered immunoblast- and centroblast-like cells . ClinicaHochheim was the first to review the literature on the subject of conjunctival lymphoma, in 1900 , 9. He rIn his descriptive paper for the Lancet in 1929, Wright was the first to make the now ubiquitous comparison of the clinical colouring of the conjunctival lymphomas to a common fish: “..lymphomatous masses under the bulbar conjunctiva, rather resembling pieces of smoked salmon, which extended backwards into the orbit over the muscle insertions” when comparing his cases to those of Bedell—the first to describe conjunctival lymphoma in the USA  were addressed in the development of a TNM-based classification system of these tumours published in 2009 . This us Edition , 23.These modern classifications stratify disease based on extent of regional nodal and distant systemic involvement at the time of diagnosis. Most conjunctival lymphomas (up to 90%) present as primary and well-circumscribed disease without evidence of prior or concurrent systemic disease , 24, 25.Pubmed (January 1964 to end of October 2021) and Medline (January 1946 to end of October 2021) databases were searched using the search terms conjunctival lymphoma and ocular lymphoma. Bibliographies were reviewed, and further papers were sourced dating back to 1872 for historic reference. Removing duplicate articles, excluding articles that were related to intraocular or orbital lymphoma, and removing most foreign language papers provided a total of 110 unique articles which were consulted and reviewed.The worldwide incidence of OAL is approximately 0.2 per 100,000, and is seen in 1–2% of all NHL and 5–10% of all extranodal lymphomas , 28. TheKirkegaard and co-workers recently performed a major review of 1014 conjunctival lymphoid neoplasms, and 98% were found to be of B-cell lineage . They foGeographically, in a multicentre study, EMZL was found to be more common in the United States and India than the United Kingdom—potentially due to a selection bias. There was also a male predominance of EMZL and FL in India, consistent with previous reports .Current understanding of conjunctival lymphoma in paediatric populations (≤18 years) is limited to case reports and small case series’ –34. In tIn a report of benign and malignant conjunctival lesions in children, Shields and co-workers reported that lymphomas had a statistically significantly larger basal diameter, and locations of diffuse (rather than bulbar), superior , inferior  and diffuse  compared to RLH . Thus, aA single case report of the behaviour of conjunctival EMZL in a pregnant woman was published in 2017 . There wHelicobacter pylori, Hepatitis C and Chlamydia spp. have been proposed to play a role in the development of EMZL . Th. Th69]. Full blood and differential counts,Biochemistry, including renal and liver function tests,Protein electrophoresis,2 microglobulin (B2M),Lactate Dehydrogenase (LDH) and βSerology for human immunodeficiency virus (HIV), Hepatitis B virus (HBV) and Hepatitis C virus (HCV).Initial staging for all extranodal lymphoma subtypes is often performed in conjunctival with a physician or haematologist. Serological testing is summarised in Table Unilateral bone marrow aspirate (with morphology and flow cytometry) and biopsy is performed to confirm suspected stage I or II disease in all patients when local treatment is planned. Some limit these investigations to select patients with multifocal disease .Formalin-fixed, paraffin embedded tissue sections are stained with haemotoxylin and eosin (H&E) to evaluate tumour morphology, and immunohistochemical (IHC) stains with antibodies directed against CD3, CD5, CD20 and CD79α are used to differentiate between T and B cell lineage. Additional IHC is used to further characterise the immunophenotype . Monocytoid cells with abundant pale cytoplasm are observed less frequently in EMZL of the conjunctiva, than other sites . PlasmacThe main differential of conjunctival MALT lymphoma, i.e., RLH, presents histologically as a polymorphous infiltrate composed of mature well-differentiated lymphocytes, often admixed with plasma cells that contain benign reactive lymphoid follicles or germinal centres. The reactive lymphoid follicles and the surrounding mantle zone contain CD20+ B lymphocytes, and CD3+ T cells predominate in the interfollicular zone. A monoclonal B cell population can be excluded by IHC, flow cytometry or IgH-PCR.de novo, or may be the result of transformation from a less aggressive subtype, most commonly FL. They show diffuse effacement of normal histological architecture and are composed of large non-cohesive cells. There are large vesicular nuclei with multiple irregular eosinophilic nucleoli. CD20 is an important B cell marker, however this can be negative after treatment with Rituximab, and in such cases CD79a or PAX5 should be employed. DLBCL can be divided into at least three immunophenotypic subgroups, which correlate with the molecular subtypes – germinal centre (GCB), activated B-cell (ABC), primary mediastinal or those unable to be classified (up to 30%) [DLBCL may develop  to 30%) .Follicular lymphoma typically demonstrates densely packed multinodular follicles that lack tingible body macrophages. The follicular population  are uniformly atypical. In general, the prognosis worsens with greater proportion of centroblasts (per high power field).MCL has a cytological similarity to EMZL, with a monomorphous B cell population closely resembling centrocytes. Aberrant CD5 co-expression by the B cells and cyclin D1 nuclear staining is important to differentiate these tumours from EMZL. The high frequency of systemic disease in these patients is also atypical for EMZL.Non-B cell lymphomas, derived from natural killer cells or T lymphocytes are uncommon and varied . They diA useful adjunct modality for demonstration of monoclonality and immunophenotype is flow cytometry. This testing requires fresh tissue. Although IHC demonstration of light-chain (κ and λ) restriction using paraffin embedded tissue can be used, it is not always reliable - as detection of cytoplasmic light chains in nonplasmacytic proliferations is prone to false-negative testing as light chains are labile and can be destroyed during routine tissue processing. Fresh tissue may also be useful for microbiological analysis, biochemical analysis and/or gene expression profiling.Morphology and immunophenotyping described above remain the gold standard in diagnosis of conjunctival lymphoma, although there is an increasing repertoire of molecular tests available to enable future targeted therapies.The molecular processes involved in EMZL and DLBCL development are complex and our knowledge thereof is constantly evolving as we apply improved molecular analyses. The processes include chromosomal translocations, mutations caused by aberrant somatic hypermutation, sporadic somatic mutations, and copy number alterations, characterised by deletions and amplifications . EMZL haTNFAIP3) gene inactivation has been shown to be associated with poor lymphoma-free survival [Most recurrent chromosomal aberrations affect or target genes encoding for regulators of NK-κB. This gene is activated by the master transcription factor NF-κB which mediates lymphocyte development, activation and survival for regulated immune responses. Genomic profiling has also shown that in ocular adnexal EMZL, the A20 gene (NK-κB inhibitor) is inactivated by somatic 6q23.3 deletion and/or mutation in up to 30% of cases , 79. Thisurvival . Toll-lisurvival . Other ksurvival , 78. TheMultiple genetic aberrations are required for the development of FL, but the genetic hallmark has been recognised as t , 18  according to the AJCC criteria. In a recent large multicentre study, stage 1E patients treated with EBRT monotherapy had a better survival than those treated with chemotherapy . Externa‘Ultra-low dose’ (or “boom-boom”) radiation therapy has been used for low-grade systemic lymphomas in recent years. Fasola and colleagues were the first to apply this to OALs in 2013, whereby patients were administered 4 Gy delivered in two 2-Gy fractions on two consecutive days . In a stDespite these promising findings, there is recent level 1 evidence demonstrating superior long-term control with 24 Gy EBRT over ultra-low-dose 4 Gy EBRT in extraorbital sites . JacksonAlthough there are few complications with ultra-low dose EBRT, early minor complications of low dose EBRT include eyelid irritation and mild conjunctivitis. Long-term complications, which occur in up to 50% of patients with doses greater than 20 Gy, include dry eye syndrome, cataract formation, retinopathy, orbital fat atrophy, and keratitis , 91.Brachytherapy can deliver high doses of radiation to target tissues, while minimising collateral exposure to surrounding healthy tissues. The use of a bidirectional Strontium-90-yttrium-90 applicator was reported in a small series by Regueiro et al. . Local cIn the setting of conjunctival disease with evidence of lymphoma at other sites, systemic chemotherapy is often indicated. The standard single-agent chemotherapy for non-Hodgkin’s lymphoma remains chlorambucil, whereas combination therapy used includes cyclophosphamide, vincristine and prednisone (CVP), and cyclophosphamide, hydroxydaunorubicin, vincristine and prednisone (CHOP) , 91. Fur2 weekly (for 4 weeks) in 2 patients with orbital disease, with a 50% complete response rate [Rituximab, a recombinant antichimeric CD20 monoclonal antibody has been used as a first line agent for cases of indolent B cell lymphoma in the ocular adnexa. Nuckel and co-workers were the first to employ rituximab at an intravenous dose of 375 mg/mnse rate . Similarnse rate , 94. In nse rate . Rituximnse rate , 95. In nse rate . There wnse rate .Proponents of first-line chemotherapy state that this treatment option eliminates local complications from radiotherapy . AlthougAs seen in other organ-specific disease, local therapies are better tolerated than intravenous and oral chemotherapies, with the latter often associated with anaemia, neutropaenia, parasthesia, hepatotoxicity, constipation and hyperglycaemia. Given the relative indolence of localised conjunctival lymphoma, compared to its systemic counterpart, clinicians have sought local therapies with more favourable side effect profiles.There are very few reports of local rituximab use in conjunctival EMZL. The largest series to date was published in 2020 by Demirci and coauthors . After dHuman interferons are a multigene family of inducible cytokines capable of exerting antitumour effects. In addition to enhancing the adaptive antitumour immune response, they can increase the expression of the tumour suppressor gene p53, inhibit angiogenesis and prime apoptosis in tumour cells . The majLocal immunotherapy with interferon-α2b is another treatment option for conjunctival EMZL, first described in 1996 . SubconjChlamydia psittaci, the routine use of topical or systemic antibiotic therapy as a first line regimen is not recommended. Doxycycline 100 mg twice daily has been shown to be a viable treatment option for T1N0M0 lymphoma with a 5-year progression free survival of 55%, where C. psittaci was confirmed [C. psittaci swabs, but there is no definitive evidence for routine use, particularly in geographical areas with low baseline infection rates.Although antibiotic therapy has been established as the standard of care in patients with gastric MALT lymphoma, much less is known about the value of antibiotic therapy in extragastric MALT. Given the controversial link between EMZL and onfirmed . A revieonfirmed . A threeClarithromycin is a substrate of P-glycoprotein which can induce apoptotic changes in tumour cells, and has been shown to exhibit antitumour activity in different murine cancer models. There has been only one report to date describing 6 months of oral Clarithromycin (500 mg twice daily) in relapsed or refractory conjunctival EMZL . Eleven Cryotherapy is an effective modality, which continues to see use in the management of primary acquired melanosis, conjunctival melanoma, conjunctival epithelial neoplasia and squamous cell carcinoma. The use of cryotherapy in conjunctival lymphoma is not well established. In cases of local resection, Shields and co-workers advocate the use of adjuvant cryotherapy . A singlThe prognosis for conjunctival lymphoma is universally good, with an overall 5-year survival of up to 94%, depending on the histological subtype, TNM stage at diagnosis and patient age , 106. ThIn a recent retrospective review of 140 patients with OAL, the 8th edition AJCC criteria were validated, with the histological subtype, lymph node involvement (≥N1), extraorbital involvement (M1) confirmed as conferring increased risk of relapse, as well as tumour diameter >30 mm and Ki-67 index >10% .It is well established that patients with elevated LDH or concurrent hepatitis C infection have also shown to have poor rates of progression-free survival . The proBilaterality continues to be debated as a risk for disseminated disease. A study of 117 patients by Shields and co-workers found that 17% of patients with unilateral conjunctival lymphoma had systemic lymphoma at the time of diagnosis, increasing to 47% in the context of bilateral conjunctival disease at presentation . As expeConjunctival lymphoma commonly affects elderly patients, although some subtypes, such as EMZL can occur in younger populations. Gender distribution varies according to lymphoma subtype, with EMZL and FL showing a female predominance. In most situations, conjunctival lymphoma is asymptomatic, but patients may present complaining of redness or ocular surface irritation. Histologic subtype and clinical stage of lymphoma are the best indicators of prognosis and patient outcome.Each lymphoma subgroup has a unique clinical course and requires tailored management with a multidisciplinary team. Local treatment administration is favourable in low grade lymphomas, with chemotherapy and/or radiotherapy reserved for higher grade lymphomas. The pathogenesis of conjunctival lymphoma is still not fully understood, but with the rapid advances in molecular analysis and immunophenotypic, we expect treatment modalities to improve and be tailored to patient’s specific presentations – with further improvement in disease-free survival.


Objective: This study investigated to probe ferroptosis-related diagnostic biomarkers and underlying molecular mechanisms in Diabetic nephropathy (DN).Methods: GSE30122 and GSE1009 from GEO database were used as training and verification sets, respectively, to screen differentially expressed ferroptosis-related genes (FRGs). These genes were further analyzed using GO, KEGG, and GSEA methods, and screened with PPI, LASSO, and SVM-RFE to identify ferroptosis-related diagnostic biomarkers for DN. A diagnostic model was established using the Glm function and verified with ROC curve. The relationship between these biomarkers and immune cell was analyzed, and qRT-PCR and Western blot were used to detect the expression of these biomarkers in kidney tissues and identify the effect of TP53 on DN development.Results: Fifty one differentially expressed FRGs were enriched in bioprocesses such as p53 signaling pathway, oxidative stress and chemical stress response, and mTOR signaling pathway. TP53, RB1, NF2, RRM2, PRDX1, and CDC25A were identified as ferroptosis-related diagnostic biomarkers for DN. TP53 showed the most differential expression. ROC analysis showed that AUC values of TP53, RB1, NF2, RRM2, PRDX1, and CDC25A were 0.751, 0.705, 0.725, 0.882, 0.691, and 0.675, respectively. The AUC value of DN diagnosis model was 0.939 in training set and 1.000 in verification set. qRT-PCR results confirmed significant differences in these six biomarkers between DN and normal kidney tissue (p < 0.05), and correlation analysis showed that five biomarkers were significantly correlated with infiltrating immune cells (p < 0.05). Furthermore, western blots showed that TP53 promotes apoptosis through PI3K-AKT signaling in DN.Conclusion:TP53, RB1, NF2, RRM2, PRDX1, and CDC25A have potential as diagnostic biomarkers for DN. The diagnostic model containing the above six biomarkers performs well in the diagnosis of DN. Five of the six biomarkers are strongly associated with several infiltrating immune cells. TP53 may play an essential role in the development of DN. Diabetic nephropathy (DN) is a severe complication associated with diabetes and the primary contributor to kidney failure . It is cItgb2 contributed to DN by promoting the transcription of EST1. HMOX1 as a potential diagnostic biomarker of atherosclerosis using bioinformatics analysis. However, few people have explored the ferroptosis-related diagnostic biomarkers of DN through bioinformatics analysis.Ferroptosis is a form of cell death, has gained significant attention in recent years due to its involvement in various pathological conditions, including DN . The iniThis study has focused on the identification of diagnostic biomarkers associated with ferroptosis and the elucidation of molecular mechanisms that drive the progression of DN. These investigations have enhanced our comprehension of the role played by ferroptosis in DN development, thus establishing a novel theoretical foundation for the treatment of DN.https://www.ncbi.nlm.nih.gov/geo) served as the primary resource for obtaining microarray datasets pertaining to diabetic nephropathy. This comprehensive database offers a vast collection of freely accessible microarray, RNA-seq, and other pertinent data, making it an invaluable tool in the domains of genetics and bioinformatics. By employing the advanced search functionality, we identified microarray profiles that included the keywords “diabetic nephropathy” and “Homo sapiens” within their titles or abstracts. Among them, the GSE30122 dataset consisted of 50 normal kidney tissue samples (control group) and 19 DN kidney tissue samples (DN group), while the GSE1009 dataset consisted of 3 normal kidney tissue samples (control group) and 3 DN kidney tissue samples (DN group). Subsequently, we downloaded the GSE30122 and GSE1009 datasets from the GEO database utilizing the “GEOquery” package. The GSE30122 dataset serves as the training set, while the GSE1009 dataset functions as the validation set. The FRGs were obtained from FerrDb (http://www.zhounan.org/ferrdb) database.The Gene Expression Omnibus (GEO) database  was set at |log2FC| > 1 and p < 0.05 were considered statistically significant.GO and KEGG pathway enrichment analyses were performed on the differentially expressed FRGs using the clusterProfiler package. Enrichment analyses with http://string-db.org). We used Cytoscape (v 3.9.1) to visualize the resulting network, and identified hub genes through plugins in Cytoscape.To identify the key significant FRGs with differential expression, we constructed a PPI network based on the STRING database  supplemented with 10% fetal bovine serum  and 100 U/mL penicillin mixture  at 37°C and 5% COTP53-silenced small interfering RNA (siRNA) and corresponding negative control were purchased from Guangzhou Ruibo Biotechnology . Lipofectamine™ 2000 was used to transfect the corresponding plasmid or siRNA into human podocyte cell line.Plasmid directly against Protein extraction was performed using RIPA buffer  supplemented with PMSF . The primary antibody  was incup < 0.05 was considered statistically significant.Data were statistically analyzed by R 4.2.1 or GraphPad Prism 9.0. Unpaired t-test was used for comparison between the two groups. ROC analysis was applied to evaluate the diagnostic ability of a single biomarker or model. Correlation analysis was performed by spearman correlation. The GSE30122 dataset was downloaded from the GEO database, and its normalization was shown in p53 signaling pathway, and mTOR signaling pathway  in the training set, and an AUC of 1.000 (95% CI: 1.000–1.000) in the validation set  and negatively regulates the transcription of Bcl-2 (anti-apoptotic protein) . In the kidney of DN patients, the mTOR signaling pathway was inhibited and subsequently suppressed the autophagy, caused mesangial expansion and proliferation, eventually led to glomerular hypertrophy and kidney damage  . Ren et y damage .P53, RB1, NF2, RRM2, PRDX1, and CDC25A. In order to validate the accuracy of the diagnostic models, we applied GSE1009 and used ROC curve analysis. The AUC value of the diagnostic model in the training set was 0.939 in the GSE30122, and the AUC of 1.000 in the GSE1009, indicating a promising predictive performance of the DN diagnostic model.PPI network was conducted on 51 FRGs and screened 10 hub genes. Next, we identified six diagnostic biomarkers for DN by lasso regression and support vector machine analysis, including TP53 is a tumor suppressor gene and participates in a variety of biological processes. Prior research has indicated that activating the TP53 pathway can heighten vulnerability to apoptosis, particularly in the context of Type 1 Diabetes Mellitus (T1DM)  . Our qPCdiseases , and ourdiseases . We alsoTP53 is the gene with the greatest expression differences. Thus, we next explored its potential regulatory pathway in DN. The PI3K/AKT signaling pathway is one of the critical pathways involved in regulating cell growth and apoptosis. Phosphatidylinositol 3-kinase (PI3K) is the important cytokine of this pathway, which has phosphatidylinositol kinase. With the dual activity of serine/threonine protein kinase, PI3K will be converted into the second messenger PIP3 after activation, and PIP3 will then activate AKT to p-AKT. p-AKT can block the combination of Bad and Bcl-2, and inhibits the occurrence and development of apoptosis (si-TP53 group showed that the PI3K-AKT signaling pathway was activated and that hallmark apoptosis markers, PARP and caspase-3, were also activated compared to the DN group. LY294002 is a commonly used PI3K/AKT pathway inhibitor (si-TP53-mediated cell apoptosis.Among all DEGs, poptosis . Westernnhibitor . Our resTP53 promotes cell apoptosis through the PI3K-Akt pathway, ultimately leading to DN.In summary, our analysis of DN data from the GEO database using bioinformatic tools led to the identification of potential molecular targets related to ferroptosis in DN. Furthermore, we developed a 6-gene model related to ferroptosis, which demonstrated excellent predictive performance for DN. Finally, qPCR and western blot analyses confirmed that