CX3CL1) gene expression and secretion, suggesting its potential as a therapeutic agent for addressing neuroinflammation and iron dysregulation. This research provides insights into the intricate connections among VD, UPR, and iron metabolism in SH-SY5Y neuroblastoma cells, with implications for future investigations and potential therapeutic approaches in neurodegenerative diseases characterized by UPR dysregulation and iron accumulation.Vitamin D3 (VD) is crucial for various cell functions, including gene regulation, antioxidant defense, and neural health. Neurodegenerative conditions are closely linked to the unfolded protein response (UPR), a mechanism reacting to endoplasmic reticulum (ER) stress. Iron metabolism is intricately associated with UPR and neurodegeneration. This study used SH-SY5Y neuroblastoma cells to investigate the relationship between UPR, iron metabolism, and VD. Different sequences of treatments (pre- and post-treatments) were applied using VD and thapsigargin (Tg), and various methods were used for evaluation, including real-time qPCR, Western blotting, ELISA, and iron content analysis. The findings indicate that VD affects UPR pathways, cytokine release, and iron-related genes, potentially offering anti-inflammatory benefits. It also influences iron transporters and storage proteins, helping to maintain cellular iron balance. Furthermore, pro-inflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) were impacting UPR activation in cells. VD also influenced fractalkine ( Vitamin D3 (VD), also known as cholecalciferol, is an essential micronutrient that has a key function in cellular metabolism. It is crucial for managing calcium balance and supporting bone well-being ,2. RecenThe unfolded protein response (UPR) is a universally acknowledged cell mechanism regulating metabolic activities. It becomes active by the presence and accumulation of improperly folded proteins in the endoplasmic reticulum (ER), which is the cellular component overseeing protein folding and quality ,9. DistuMoreover, the UPR oversees other cellular functions like cell longevity, autophagy, and inflammatory response . It aidsIron is indispensable for the neural system, aiding neurotransmitter production, myelin creation, and brain metabolic processes ,16. DiscInflammatory cytokines like IL-1, IL-6, and TNF-α affect iron balance through the elevation of hepcidin, leading to altered iron absorption. This mechanism restricts iron during inflammation, preventing potential harm. The link between inflammatory cytokines and UPR is labyrinthine, with cytokines potentially inducing UPR pathways, thereby contributing to proper protein folding and ER stability during inflammatory conditions ,21.CX3CL1, is a unique chemokine vital for immune cell recruitment and activation by binding to the CX3CR1 receptor. It is fundamental in processes like inflammation, immune cell migration, and neuronal responses. Its role in the nervous system is paramount, affecting brain development, synaptic adaptability, and reactions to neurodegenerative diseases [PDIA3). This receptor might account for the immediate actions of VD [Fractalkine, also referred to as diseases . Importadiseases ,24,25. Vdiseases . In immuns of VD . FurtherWe aimed to explore UPR’s influence on iron balance and understand how pre- and post-administration VD might mitigate the stress caused by UPR concerning iron homeostasis.DDIT3, HSPA5, and XBP1. After the Tg treatment, the relative expression of every investigated gene increased significantly compared to the control  family and is pivotal in the facilitation of protein structuring and surveillance within the ER. Consequently, any dysregulation of PDIA3 could be linked to the initiation of the UPR. In the Tg-administered cells, the PDIA3 gene’s relative expression has increased significantly  and TNFα. The results showed a pattern in all three cases. In the Tg group, elevated cytokine concentrations were measured for all three cytokines A–C. For PDIA3. Relative expression of CX3CL1 was increased in the groups with Tg treatment alone and VD pre-treatment , VD, and iron metabolism involves intricate crosstalk among signaling pathways. UPR activation can influence the NF-κB, which is a transcription factor complex consisting of p105, a propeptide of p50 and p65 subunits, which regulates the expression of genes involved in immune responses, inflammation, and cell survival. The NF-κB pathway, such as the STAT3 pathway, can be activated by pro-inflammatory cytokines, and it has a crucial role in iron metabolism by regulating the expression of hepcidin, iron transporters, and iron storage proteins, thereby influencing iron absorption, distribution, and utilization in response to inflammatory stimuli. VD, through its interaction with the NF-κB pathway, STAT3 pathway, and UPR by the Erp57 protein, can modulate iron metabolism by regulating hepcidin expression, thereby impacting iron homeostasis. In the case of activated (phosphorylated) STAT3 (pSTAT3), a significant reduction was measured in only the VD-treated cells compared to the Tg administration alone. However, the relative expressions of p105 and p65 were increased in this group. Among the co-treatments, changes were only observed in the VD pre-treated group, with positive differences for p105 and negative differences for p65 compared to Tg-treated cells .Neurodegenerative diseases, increasingly recognized as significant social and medical burdens, are driven by complex mechanisms often involving the accumulation of proteins and iron, though their precise initiations are not always known ,31. ThisIn order to carry out our investigations, we utilized SH-SY5Y human neuroblastoma cells, a well-established model in neurological research ,33,34. TVD, or cholecalciferol, is an essential micronutrient involved in various cellular processes. It regulates gene expression of over 200 genes and has been shown to be important for cell growth, differentiation, and apoptosis ,39,40. AVD has been shown to modulate UPR signaling pathways, influencing protein folding and quality control in the central nervous system . In the PDIA3, being an important receptor of VD, exhibits distinct patterns of mRNA expression based on different treatments. In the VD+Tg group, the mRNA expression levels remained high, similar to the samples treated with Tg alone. However, in the case of VD post-treatment, there was a decrease in mRNA levels compared to the Tg group. Interestingly, the protein expression of ERp57 displayed a contrasting pattern. Both VD pre- and post-treatment led to a significant reduction in ERp57 protein expression compared to both the control and Tg groups  increased the relative expression of HAMP gene mRNA, resembling the effect observed with Tg treatment alone. However, when VD was administered after Tg treatment, the mRNA expression level of the HAMP gene returned to levels equivalent to the control group. These results suggest that VD post-treatment resolves the iron retention induced by UPR at the mRNA level.The intricate interplay between ERp57 and iron metabolism has been established via its interactions with the STAT3 and NFκB signaling pathways ,55,56,57HAMP gene and vitamin D, underscores the complexity and significance of iron regulation in health and disease.Intracellularly, iron distribution is meticulously managed across various organelles, each playing a unique role in iron homeostasis. Mitochondria are key organelles that utilize iron for essential roles, notably in synthesizing components like heme and iron-sulfur clusters, which are critical for ATP production . The cytGiven the influence of the treatment order on the key regulator of iron metabolism, we investigated the behavior of iron transporters, specifically DMT1 (responsible for iron uptake) and FPN (involved in iron export from the cell). Analysis of gene expression revealed that in the relative expression of DMT1, a statistically significant reduction was observed in the VD post-treated group compared to Tg administration alone but not in the expression of FPN A. All thTo further investigate potential changes in iron utilization, we examined the expression of FTH, a protein involved in cytosolic iron storage, at both the mRNA and protein levels. The results revealed an elevation in relative mRNA expression of FTH in the Tg and VD+Tg groups compared to the control. Conversely, VD-only treatment and VD post-treatment resulted in lower mRNA expression levels compared to Tg treatment alone. Protein analysis showed a significant decrease in FTH levels after Tg administration and VD post-treatment. Interestingly, the VD-only and VD pre-treated groups exhibited a statistically significant increase in FTH protein expression compared to Tg administration alone. Total iron content analysis was performed to assess the overall iron content within the cells. Although statistically significant differences were not observed, a similar pattern was noted to that observed with FTH levels. Previous research has investigated the interrelationships among iron status, ferritin levels, and VD concentrations ,61. The Our research delved into the involvement of cytokines in the regulation of iron metabolism and the UPR, especially in relation to neurodegenerative conditions. Our findings indicate that cytokines such as IL-6 and TNFα can modulate hepcidin expression and influence UPR activation in neurons, suggesting their potential contribution to disease pathogenesis. The measurement of secreted IL-6, IL-8, and TNFα revealed elevated cytokine concentrations in the Tg group, indicating a pro-inflammatory state. Interestingly, both pre- and post-administration of VD resulted in a significant reduction in IL-6 and IL-8 secretion, suggesting a potential anti-inflammatory effect of VD in the context of iron accumulation in neurodegenerative diseases. Pro-inflammatory cytokines significantly increase the amount of hepcidin, which contributes to the retention of iron ,63. In cInvestigation into fractalkine, a protein involved in iron metabolism and UPR, demonstrated changes in its gene expression in response to VD treatments. While VD post-treatment resulted in a reduction in fractalkine gene expression compared to Tg-only treatment, VD pre-treatment led to an increase. Additionally, VD alone was found to reduce fractalkine expression and increase its protein secretion, indicating a unique impact of VD on fractalkine regulation. Overall, the results highlight the ability of VD to modulate cytokine secretion and gene expression, along with its influence on fractalkine, suggesting its potential as a therapeutic target for managing neuroinflammation and iron dysregulation in neurodegeneration. Banerjee et al. (2018) found that VD administration resulted in a substantial reduction in the levels of fractalkine, evidenced by a 50% decrease following treatment with TNFα . This obTo our knowledge, the literature provides only a limited number of studies that probe an in-depth analysis of the interactions between VD, the UPR-inducing agent Tg, and iron metabolism. Particularly, studies exploring these relationships beyond the scope of pre-treatment, extending into post-administration phases, are notably absent. Our research aims to elucidate these complex connections, thereby contributing to a more comprehensive understanding of the physiological impact of VD in the context of UPR and iron regulation.The limitations of our study lie in the fact that they are in vitro experiments using undifferentiated SH-SY5Y cells; this study was conducted with one UPR-inducing agent, and only one type of cell was used to carry out the experiments. The generation and verification of UPR can be best studied with cell cultures. It is our plan to perform further investigations with differentiated cells to see whether they behave in another way for UPR than the undifferentiated ones. As the calcium ion’s role in neurons is not only to provide the proper storage in the ER but also to be involved in cell signaling and neuronal excitation, we will look at other ways of UPR generation and its effect on neuronal behavior. It will be an interesting question whether VD is having the same or different effect in these cases. According to the literature, on some occasions, pre-treatment of VD is more effective in some UPR-producing methods . Iron meGiven the established role of VD in calcium homeostasis, it is imperative that future analyses incorporate an examination of calcium metabolism. This inclusion will enable a more holistic understanding of VD’s biological impact and its potential interactions with other metabolic pathways.2 at 37 °C. For gene expression analysis, 4 × 105 cells were placed on a 6-well plate, while for protein analysis, 106 cells were seeded on a cell culture flask with a 25 cm2 surface area. The cells were treated with VD at a final concentration of 25 nM and/or Tg at a final concentration of 25 nM; both were dissolved in absolute ethanol. Four different treatment methods were used during the experiments as follows: VD followed by ethanol, Tg then ethanol, VD then Tg, and Tg before VD. The second agent was administered after 24 h of incubation following treatment with the first agent. The cells were collected after incubation with the second medium for 24 h.In our research, SH-SY5Y neuroblastoma cells were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12  with 10% fetal bovine serum , 1% nonessential amino acids  and 1% penicillin/streptomycin mixture  in a humid environment with 5% COTo analyze gene expression, total RNA was isolated from the collected cells with Aurum Total RNA Mini Kit  according to the manufacturer’s protocol. The extracted RNA’s concentration was determined with a MultiSkan GO spectrophotometer  using the µDrop plate . Thenceforth, complementary DNA was synthesized from 200 ng of total RNA of each sample with iScript Select cDNA Synthesis Kit . Gene expression analysis was performed by the real-time quantitative PCR method using CFX96 One Touch Real-Time PCR System  and iTaq Universal SYBR Green Reagent Mix . The Livak (∆∆Ct) method was implemented to evaluate data of relative quantification. In every sample, the gene of interest’s expression ratio was determined by normalizing its value against that of β-actin, serving as an internal standard. Throughout the analysis, the expression level of this reference gene was arbitrarily assigned a baseline value of one for comparative purposes. The sequences of primers used in the analysis are listed in w/v)) blocking solution  was used for blocking the membranes for 1 h at 25 °C with gentle shaking. Then, the membranes were incubated for 1 h at 25 °C with ferroportin (anti-FPN) IgG , the divalent metal transporter-1 (anti-DMT-1) , and the fractalkine (anti-CX3CL1) IgG  polyclonal rabbit antibodies. The ferritin heavy chain (anti-FTH) IgG , the binding immunoglobulin protein (anti-BiP) IgG , the spliced X-box-binding protein (anti-XBP-1 spliced) , the endoplasmic reticulum protein 57 (anti-Erp57) IgG  polyclonal rabbit antibodies, and the C/EBP homologous protein (anti-CHOP) IgG  polyclonal mouse antibody were overnight incubated at 4 °C. For the loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH)  was used in the Western blot protein analysis. Horseradish peroxidase (HRP)-linked goat anti-rabbit IgG was applied  as the secondary antibody, and it was incubated for 1 h at 25 °C. The blotted proteins were detected by using WesternBright ECL chemiluminescent substrate . The membranes were developed using the Alliance Q9 Advanced gel documentation system . The analysis of the protein bands’ optical density was determined by ImageJ 1.53t software [SH-SY5Y cells were collected by centrifugation after treatments. The cells were lysed with 150 µL of ice-cold lysis buffer (50 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7.4, 150 mM sodium chloride (NaCl), 0.5% Triton-X 100) containing complete mini protease inhibitor cocktail . The protein concentration was determined using a Detergent Compatible Protein Assay Kit , and an equal amount of protein was separated from each sample in 10%, 12%, or 14% polyacrylamide gels. Mini-PROTEAN Tetra Cell polyacrylamide gel electrophoresis system  was used for the vertical electrophoresis. The protein transfer from the gels was carried out by semi-dry electroblotting to nitrocellulose membranes . Non-fat dry milk  according to the manufacturer’s instructions. The absorbance measurements were performed by using a MultiSkan GO microplate spectrophotometer  at 450 nm wavelength. The intensity of the signal is directly proportional to the concentration of the target present in the samples. The concentrations of cytokines were expressed as pg/mL.3 standard curve and reported as mM iron/mg protein.After the treatments, the cells were harvested by centrifugation and were lysed in 50 mM NaOH for 2 h with gentle shaking at room temperature. An equal volume of 10 mM HCl was added to the samples to neutralize the samples. After neutralization, the samples were mixed with acidic KMnO4 and incubated at 60 °C for 2 h to release protein-bound iron. Reduction of iron and complex formation with ferrozine was carried out in the aqueous solution of 6.5 mM neocuproine, 1 M ascorbic acid, 2.5 M ammonium acetate, and 6.5 mM ferrozine at room temperature for 30 min. The purple ferrozine–iron complexes are detectable at 550 nm. Absorbance readings were obtained through a MultiSkan GO spectrophotometer . Sample concentrations were determined by reference to a FeClp-value below 0.05 denoting statistical significance. In the figures, the asterisks represent a significant difference compared to the control, while the crosses represent a significant difference compared to the Tg treatment.Statistical evaluations were performed utilizing SPSS software, version 24.0 . Once a normal distribution had been confirmed, the significance of the data was determined using a one-way ANOVA with an LSD post hoc test for detailed comparisons. The results are presented as mean ± standard deviation (SD), with a In conclusion, this study underscores the intricate relationship between the unfolded protein response, iron metabolism, and VD in neurodegenerative diseases. The research provides insights into the distinct responses of the ERp57/PDIA3 protein, an essential mediator of UPR and a receptor of VD, to various treatment regimens. Notably, VD administration was found to influence UPR activation, iron transporter dynamics, and cytokine secretion, showcasing its capacity to act as a modulator in neuroinflammatory processes and dysregulation of iron homeostasis. The findings illuminate the complex interplay between VD, UPR, and iron metabolism, providing a promising avenue for therapeutic intervention in neurodegenerative conditions characterized by UPR dysregulation and iron accumulation.


To investigate the potential mechanism of Er-Xian decoction (EXD) in treating aplastic anemia (AA), the active components of EXD were screened by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the targets of the components were predicted by the Swiss Target Prediction database. AA targets were collected from the GeneCards, OMIM, DisGeNET, PharmGKB, DrugBank, and TTD databases, the intersection of AA targets and EXD targets was calculated, and an herb-component-target network was constructed by Cytoscape 3.7.2 software. The STRING database was used for protein‒protein interaction (PPI) analysis, and Cytoscape 3.7.2 software was used to construct a PPI network and perform topology analysis. The core targets were imported into the DAVID database for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The molecular docking software AutoDock was used to measure the affinity between active components and key targets. Finally, we established a mouse model of AA and verified the key targets and signaling pathways of EXD by RT‒PCR, ELISA and Western blot analysis. A total of 53 active components were screened from EXD, 2516 AA-related targets were collected, and 195 common targets were obtained. An herb-component-target network and a PPI network were successfully constructed, and 36 core targets were selected from the PPI network. The main active components of EXD include luteolin, kaempferol, berberine, etc., and key targets include PIK3CA, AKT1, STAT3, etc. GO functional enrichment analysis showed that cell components, molecular functions and biological processes with significant correlations were macromolecular complexes, protein serine/threonine/tyrosine kinase activity and protein phosphorylation, respectively. KEGG pathway analysis showed that the pathways with significant correlations included the PI3K-Akt signaling pathway and JAK-STAT signaling pathway. Molecular docking results showed that the tested key targets had good affinity for the corresponding active components. In AA mice, we found that EXD significantly increased white blood cell count, red blood cell count, platelet count and hemoglobin levels, increased mRNA levels of PIK3CA, PIK3CD, AKT1, JAK2, STAT3 and MAPK1, and promoted phosphorylation of PI3K, AKT, ERK1/2 and STAT3. In summary, EXD acts on PI3K, AKT, STAT3 and other targets through berberine, luteolin, quercetin and other components to regulate the PI3K-Akt pathway, JAK-STAT pathway and other pathways, thus exerting its therapeutic effect on AA. This study explained the Chinese medicine theory of treating AA with EXD by tonifying kidney-yang and provides a scientific basis for the use of EXD in treating AA. The main clinical manifestations consist of different degrees of anemia, hemorrhage and infection with significant morbidity and mortality2. At present, the pathogenesis of AA has not been fully elucidated, and it is generally believed that AA is related to hyperimmune function, a defective hematopoietic microenvironment, a shortage or dysfunction of hematopoietic stem cells, and genetic abnormalities3. The incidence of AA is significantly higher in East Asia than in other regions, especially among young people4. AA can be divided into very severe aplastic anemia (VSAA), severe aplastic anemia (SAA), and nonsevere aplastic anemia (NSAA) according to severity in the Camitta standard and into acute AA and chronic AA according to the progression of the disease in China6. For young patients with immune AA, bone marrow transplantation is the preferred treatment, which can achieve satisfactory results8. Immunosuppressive therapy is appropriate for all patients, especially those who are not candidates for bone marrow transplantation, but late consequences of the disease may occur because it cannot replace the affected bone marrow or immune system1. Other treatment strategies include androgen and stem cell stimulation, but the results are often less than satisfactory. Therefore, searching for an alternative and more effective treatment of AA is necessary.Aplastic anemia (AA) is a rare immune-mediated hematopoietic disorder characterized by weakened proliferation of bone marrow hematopoietic cells, reduced peripheral whole blood cells, and immune dysfunction10. There are two views on the pathogenesis of AA in Chinese medicine: deficiency-caused AA (Yin-Xu-Zhi-Bing in Chinese) and poison-caused AA (Yin-Du-Zhi-Bing in Chinese), which correspond to immune AA and acquired AA in general9. The theory of deficiency-caused AA posits that deficiency and damage to kidney essence leads to depression of kidney Yang (Shen-Yang-Xu in Chinese), which cannot stimulate bone marrow hematopoiesis, resulting in blood deficiency11. Chinese medicine posits that the kidney dominates the bone and generates the marrow (Zhugu-Shengsui in Chinese), so tonifying the kidney and warming the kidney Yang is the fundamental strategy to treat AA11.In traditional Chinese medicine (TCM) theory, there is no corresponding disease name of AA, which can be classified into the category of “deficiency and strain” (Xulao in Chinese), “bone strain” (Gulao in Chinese), “blood deficiency” (Xuexu in Chinese), and “blood depletion” (Xueku in Chinese) according to its clinical manifestations, and it is thought that the lesion is mainly in the bone marrow, involving the spleen, kidney and liver12. EXD possesses the effect of warming kidney Yang (Wen-Shen-Yang in Chinese) and tonifying kidney essence (Bu-Shen-Jing in Chinese) and is widely used in the clinical treatment of menopausal syndrome13. Modern scientific studies have shown that EXD also has many therapeutic effects, such as anti-osteoporosis, ameliorating myocardial damage during menopause, repairing spinal cord injury and anti-depressant effects17. Based on the kidney-tonifying effect of EXD and the traditional Chinese medicine theory of tonifying the kidney in the treatment of AA, we believe that EXD can be used to treat AA. In fact, clinical studies have shown that EXD has a significant curative effect on AA. A clinical study of 84 AA patients treated with EXD showed that the total effective rate of EXD could reach 84.5%. Another study showed that the use of EXD in addition to cyclosporine and stanozolol significantly improved bone marrow hematopoietic function and reduced complications19. Some herbs or ingredients in EXD have also been used alone to improve AA pathology; for example, DG polysaccharide prevents mitochondrial apoptosis by regulating the Treg/Th17 ratio in aplastic anemia, and icariin in YYH can improve the hematopoietic function of cyclophosphamide-induced myelosuppression mice21. In the current study, we observed the therapeutic effect of EXD on AA mice and explored the mechanism using network pharmacology combined with experimental validation.Er-Xian decoction (EXD) is a classic TCM formulation comprising 6 herbs: Rhizoma Curculiginis , Epimedii Folium , Radix Angelicae Sinensis , Radix Morindae Officinalis , Cortex Phellodendri Chinensis , and Rhizoma Anemarrhenae https://old.tcmsp-e.com/tcmsp.php) was used to search the chemical ingredients of XM, YYH, DG, BJT, HB, and ZM in EXD22. The active ingredients were screened according to oral bioavailability (OB) > 30% and drug-likeness (DL) > 0.18, and the ingredients without PubChem CID were removed. All active ingredients were queried canonical SMILES by the PubChem database (https://pubchem.ncbi.nlm.nih.gov), and then the putative targets were predicted by the Swiss Target Prediction database according to the canonical SMILES.The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  using “aplastic anemia” as a key wordhttps://bioinfogp.cnb.csic.es/tools/venny), which represents potential targets for EXD treatment of AA. The component-target network was constructed using active components and potential targets by Cytoscape 3.7.2 software29.The intersection of AA targets and EXD targets was identified by the Venny2.1 platform (https://cn.string-db.org), the organism was set as “Homo sapiens”, the minimum required interaction score was set as 0.7, other parameters were set as default, and PPI analysis was performed30. Topology analysis was used to measure the importance of nodes, including three parameters: degree, betweenness, and closeness.The potential targets of EXD for AA were imported into the STRING database (https://david.ncifcrf.gov)31. In brief, the gene symbols of potential targets were imported into DAVID, the organism was set as “Homo sapiens”, and GO functional and KEGG pathway enrichment analyses were performed33.To reveal the multiple mechanisms of EXD treatment of AA, potential targets were used for GO functional and KEGG pathway enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery , and the structure of the active compound was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). After pretreatment by PyMOL software or Chem3D software, the active compound and the target were imported into AutoDock software for docking. The Lamarckian genetic algorithm (LGA) was used to find the best binding state between the ligand (active compound) and receptor (target), and energy matching was used to evaluate the binding ability between the ligand and receptor.Virtual molecular docking was employed to evaluate the affinity between the active compounds and the key targets of EXD in the treatment of AA. The structure of the target was obtained from the RCSB Protein Data Bank  was ground into a powdered form. The constituents were extracted twice with 5 L of double-distilled water at 100 °C for 0.5 h each time. Then, the mixed extract was filtered and concentrated at 50 °C, lyophilized to yield EXD powder, and then stored at − 20 °C for subsequent use. By calculation, the extraction rate of EXD from herbs is approximately 20.31%.All 6 kinds of Chinese herbs in EXD were purchased from Sichuan Chinese herbal medicine Co., LTD  and identified as genuine by Professor Guangxian Liu of the Affiliated Hospital of Panzhihua University. The EXD was prepared by improving the previous methodMale and female BALB/c mice (6 weeks old and weighing 18–22 g) were purchased from Chengdu Dossy Experimental Animal Co., LTD [license number: SCXK (Chuan) 2015–030] . Mice were housed under specific pathogen-free conditions and cared for in accordance with the guidelines of the National Science and Technology Committee of China. All procedures and animal experiments were approved by the Animal Care and Use Committee of Panzhihua University.34. Briefly, the mice were exposed to 6.0 Gy total body irradiation at approximately 1.0 Gy/min and injected via the tail vein with two million lymphocytes isolated from the lymph glands of DBA mice within 4 h after irradiation. Seventy-two hours after irradiation, intragastric administration was started, the normal group and AA group were given normal saline, and the EXDL, EXDM, and EXDH groups were given the corresponding doses of EXD 16. All animals were treated continuously for 21 days. All mice were killed by cervical dislocation after treatment.A total of 50 mice were randomly divided equally into five groups : normal, AA, low-dose EXD (EXDL), medium-dose EXD (EXDM), and high-dose EXD (EXDH). Except for the normal group, all mice in the other groups were used to establish the AA model according to a previous methodAfter treatments, peripheral blood was collected from the fundus venous plexus of mice for routine analysis, including white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT) and hemoglobin (HB).After the mice were sacrificed, the femur was removed, and normal saline was used with a syringe to flush out the bone marrow cells for PCR analysis. Total RNA was isolated from bone marrow cells using an RNAsimple Total RNA Kit  according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA using a HiScript II 1st Strand cDNA Synthesis Kit , and then AceQ Universal SYBR qPCR Master Mix  was used to perform real-time PCR using cDNA as a template. Using GAPDH as a reference gene, the relative expression level of the target gene was calculated according to the comparative Ct method. The primers used in the experiment were as follows: forward sequence CACCTGAACAGACAAGTAGAGGC and reverse sequence GCAAAGCATCCATGAAGTCTGGC for PI3KCA, forward sequence ACCATCAGTGGCTCTGCGGTTT and reverse sequence GTGGTCTTCTGGGAACTCACCT for PI3KCD, forward sequence GGACTACTTGCACTCCGAGAAG and reverse sequence CATAGTGGCACCGTCCTTGATC for AKT1, forward sequence GCTACCAGATGGAAACTGTGCG and reverse sequence GCCTCTGTAATGTTGGTGAGATC for JAK2, forward sequence TCAAGCCTTCCAACCTCCTGCT and reverse sequence AGCTCTGTACCAACGTGTGGCT for MAPK1, forward sequence AGGAGTCTAACAACGGCAGCCT and reverse sequence GTGGTACACCTCAGTCTCGAAG for STAT3, and forward sequence CATCACTGCCACCCAGAAGACTG and reverse sequence ATGCCAGTGAGCTTCCCGTTCAG for GAPDH.35. Briefly, bone marrow cells were lysed on ice with RIPA solution for 20 min and then centrifuged at 12,000 r/min at 4 °C for 10 min. The supernatant was mixed with 5 × loading buffer, heated at 95 °C for 5 min, shock cooled and mixed for SDS gel electrophoresis. Then, the protein in the gel was transferred to a PVDF membrane and blocked with 5% skim milk TBST solution at room temperature for 1 h. It should be noted that before membrane transfer, the PVDF membrane of appropriate size only covered the gel of the target protein region, so the blot image cannot show the complete gel. The 5% skim milk TBST solution was discarded, and the membrane was washed 3 times with TBST. Then, the primary antibodies, including p-PI3K, PI3K, p-AKT, AKT, p-STAT3, STAT3, p-ERK1/2, ERK1/2, and β-actin , were added and incubated at 4 °C overnight. The next day, the primary antibody was discarded and washed 3 times with TBST, and the secondary antibody was added and incubated at room temperature for 1 h. An ECL chemiluminescent reagent  was used to develop protein bands on the membrane. These bands were quantitatively analyzed by ImageJ software .Western blot assays were carried out according to previously described methodsP < 0.05 was considered statistically significant.The statistical analysis in this study included two aspects: the P values of the GO and KEGG enrichment analyses in the network pharmacological analysis were completed by the DAVID database, and the data obtained from the experiment were expressed as the mean ± standard deviation (SD). GraphPad Prism 8.0 software  was used for statistical analysis and mapping. For experimental data, significant differences between groups were assessed using one-way ANOVA followed by Tukey’s multiple comparison test or two-way ANOVA followed by Sidak’s multiple comparisons test. We state that all experiments in this study follow the guidelines for ARRIVE and comply with ethical requirements. The animal experiments in this study followed the Guidelines for Ethical Animal Welfare (2018 edition) of the National Science and Technology Committee of China and were approved by the Experimental Animal Welfare Ethics Committee of the Affiliated Hospital of Panzhihua University.According to the established screening criteria, 4 ingredients were collected from XM, 14 from YYH, 2 from DG, 12 from BJT, 23 from HB, and 9 from ZM Table . Beta-siTo explore the interactions between 195 potential targets, we imported these targets into the STRING database for PPI analysis and imported PPI data into Cytoscape software to build a PPI network. The PPI network has 179 nodes and 848 edges, representing 179 potential targets and 848 pairs of interaction relations, respectively. The larger the area of the node and the darker the color, the larger the degree value of the node and the more important it is Fig. A. Under Homo sapiens for GO functional and KEGG pathway enrichment analyses, including cellular component (CC), molecular function (MF), and biological process (BP). A total of 343 GO terms with P < 0.05 were enriched, including 33 CC terms, 58 MF terms, and 252 BP terms. We selected the top 10 terms for significance, as shown in Fig. P < 0.05, and Fig. The 36 core targets were used for GO functional and KEGG pathway enrichment analysis. In brief, the gene symbols of 36 core targets were imported into the DAVID database, and the species was set as Molecular docking was used to simulate the binding between the active ingredients of EXD and the key targets. Combined with the results of PPI network analysis and target-pathway network analysis, we selected PI3Kα (PIK3CA), AKT1, ERK2 (MAPK1) and STAT3 as the tested macromolecules to examine their binding energy with their corresponding active compounds. The results showed that coumaroyltyramine can form hydrogen bonds with LEU-755, HIS-670 and TYR-836 residues of PI3Kα, and the binding energy was − 4.82 kcal/mol; luteolin can form hydrogen bonds with GLN-79 and THR-211 residues of AKT1, and the binding energy was − 5.76 kcal/mol; and cavidine can form hydrogen bonds with ARG-77 and TYR-139 residues of ERK2, and the binding energy was − 6.07 kcal/mol -9,10-anthraquinone, and cavidine. These 10 compounds acted on 109 of 195 potential targets, suggesting that these compounds may play an important role in the treatment of AA. Anti-inflammatory and antibacterial effects of 1,5,15-tri-O-methylmorindol and 1,6-dihydroxy-5-methoxy-2-(methoxymethyl)-9,10-anthraquinone have been reported, but more pharmacological activities have not been fully studied39. As a well-known natural product, berberine has a wide range of pharmacological activities, such as antibacterial, anti-inflammatory, antitumor, blood pressure regulation, metabolic regulation and neuroprotective effects41. Berberine can inhibit T helper cell activity by interfering with cytokine receptors or downstream signaling pathways42. This suggests that berberine weakens the immune system’s attack on hematopoietic cells through immunosuppression, which may contribute to the treatment of AA. Luteolin, quercetin and kaempferol are very common flavonoids that share similar chemical structures and have a wide range of biological activities, such as anti-inflammatory, antitumor and neuroprotective properties. Other studies have reported that oral gavage administration of luteolin alleviates anemia in HgCl2-treated rats, luteolin 7-glucoside guides hematopoietic stem cell differentiation into the erythroid lineage, and quercetin has a potential therapeutic effect on Fanconi anemia and inhibits protein and lipid oxidation on the erythrocyte membrane in Leishmania-infected animals, thereby arresting the development of anemia during the postinfection period46. Cavidine alleviates LPS-induced organ damage through antioxidant and anti-inflammatory activities48. The potential benefits of these compounds on hematopoietic function and inhibitory potential on immune function partly reflect the material basis of EXD treatment of AA.EXD is a prescription for tonifying the kidney and warming Yang and is widely used for diseases related to deficiency of kidney Yin and Yang52. GO and KEGG analyses showed that 252 biological processes and 122 signaling pathways were involved in the treatment of AA by EXD. GO functional analysis reflected that the most significant biological process was protein phosphorylation, the most significant cell component was macromolecular complex, cytosol, and cytoplasm, and the most significant molecular function was protein serine/threonine/tyrosine kinase activity. These results suggest that the mechanism of EXD treatment of AA is mainly related to the massive signal transmission occurring in the cytoplasm. KEGG pathway enrichment analysis showed that the PI3K/AKT signaling pathway was the most important pathway, and the JAK/STAT signaling pathway and MAPK signaling pathway were also enriched. Additionally, the core target-pathway network further reveals the importance of PI3K, AKT and MAPK. One of the most important signal transduction mechanisms in hematopoietic cells is the production of the lipid second messenger PIP3 regulated by PI3K53. PI3K converts PIP2 to PIP3, which leads to activation of the serine/threonine kinase AKT54. Activation of AKT promotes activation of downstream signals, which regulate the cell cycle, apoptosis, protein translation, autophagy, and metabolism55. Reduced PIP3 signaling impairs most aspects of hematopoiesis, including hematopoietic stem cell homeostasis and the development or function of T, B, and NK cells, myeloid mast cells, monocytes, granulocytes, and erythrocytes56. Therefore, activating the PI3K/AKT signaling pathway has positive significance for promoting hematopoietic development. The JAK/STAT pathway is another important signal regulating hematopoietic development. STAT3 is the only STAT family member that is essential for development; it plays a central role in the regulation of hematopoiesis and plays a critical role in the development of T helper cell and B-cell subsets as well as dendritic cell development and maturation57. Cytokines activate or inhibit STAT3 activity in hematopoietic progenitors to maintain moderate hematopoietic levels; however, hyperactivation or mutation of STAT3 can lead to tumor development59. ERK1  and ERK2  play crucial roles in cell survival, proliferation, migration, and differentiation in many tissues. Inhibition of ERK activation has been shown to hinder the transition of hematopoietic progenitor cells, and the absence of ERK1 and ERK2 in murine hematopoietic cells leads to bone marrow aplasia, leukopenia, anemia, and early lethality61.We conducted PPI analysis of potential targets of EXD for AA treatment and found that AKT1, STAT3, EGFR, TP53, BCL2, JAK2 and STAT1 were the most important nodes in the PPI network after two screenings, suggesting that these seven genes play an important role in the treatment of AA by EXD. Previous studies have demonstrated that the PI3K/AKT signaling pathway, EGFR/ERK signaling pathway, and JAK-STAT signaling pathway promote hematopoietic cell proliferationTo preliminarily verify the affinity between the active ingredients of EXD and the key targets, we simulated the binding situation by the molecular docking method, and the results showed that the tested compounds had good affinity with the target. To further verify the analysis results, we conducted experiments on an AA mouse model. The results showed that EXD significantly improved the peripheral blood cell count of AA mice, activated the PI3K/AKT pathway, and promoted the phosphorylation of STAT3 and ERK1/2. These data confirm the results of our network pharmacological analysis.In conclusion, this study preliminarily revealed the main active ingredients and potential molecular mechanisms of EXD in the treatment of AA through network pharmacological analysis combined with animal experiments, providing a scientific basis for the use of EXD in the treatment of AA. However, this study also has some shortcomings. For example, this study collected the active ingredients of various herbs in EXD through a public database as the active ingredient cluster of EXD, which may be different from the real pharmacodynamic substances in EXD. In addition, the target prediction of active ingredients depends on the algorithm of the database, but the regulatory relationship between these components and corresponding targets has not been verified by experiments. Our study partially revealed the molecular mechanism of EXD treatment of AA, and a more comprehensive and in-depth mechanism needs further research.In the present study, we revealed the molecular mechanism by which EXD regulates the PI3K/AKT signaling pathway, JSK/STAT signaling pathway, and other pathways for AA treatment using network pharmacology and verified these mechanisms in an AA mouse model. This study uses modern scientific methods to clarify the effectiveness and mechanism of EXD in treating AA by warming Yang and tonifying the kidney (Wen-Yang-Bu-Shen in Chinese) and provides a scientific basis for the ethnopharmacological use of EXD.Supplementary Information.


Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron–sulfur cluster enzymes: IspG and IspH. IspG and IspH are rate limiting-enzymes of the MEP pathway but are challenging for metabolic engineering because they require iron–sulfur cluster biogenesis and an ongoing supply of reducing equivalents to function. Therefore, identifying novel alternatives to IspG and IspH has been an on-going effort to aid in metabolic engineering of terpenoid biosynthesis. We report here an analysis of the evolutionary diversity of terpenoid biosynthesis strategies as a resource for exploration of alternative terpenoid biosynthesis pathways. Using comparative genomics, we surveyed a database of 4,400 diverse bacterial species and found that some may have evolved alternatives to the first enzyme in the pathway, Dxs making it evolutionarily flexible. In contrast, we found that IspG and IspH are evolutionarily rigid because we could not identify any species that appear to have enzymatic routes that circumvent these enzymes. The ever-growing repository of sequenced bacterial genomes has great potential to provide metabolic engineers with alternative metabolic pathway solutions. With the current state of knowledge, we found that enzymes IspG and IspH are evolutionarily indispensable which informs both metabolic engineering efforts and our understanding of the evolution of terpenoid biosynthesis pathways. All terTerpenoids are currently harvested from plants, generated by microbial fermentation, or produced chemically from nonrenewable petroleum . To dateTerpenoids are used in membranes in all known domains of life  and are Of the two known modern metabolic routes that generate the five carbon building-block precursor molecules from which all terpenoids are derived, the MVA pathway is generally used by eukaryotes and archaea  and the The first enzyme of the MEP pathway, Dxs, is an initial bottleneck to carbon flux . This boMultiple routes that allow native or acquired circumvention of the first MEP pathway enzyme Dxs have been identified but to date no alternative routes circumventing IspG or IspH have been found . In contHere we define evolutionary flexibility as steps in metabolism for which multiple enzymatic solutions have evolved, as is seen in the MVA pathway . In cont2.2.1.Fasta files of the NCBI database of reference and representative bacterial genomes available at the assembly level “complete chromosome” were downloaded (access date 6/19/2023). Profile Hidden Markov Models (pHMM) from the KOfam database  were dowhttps://github.com/bamarshall2/evolutionary_flexibility.Full bioinformatic pipeline is available at 2.2.Spiroplasma, Mycoplasma, Mycoplasmoides, Malacoplasma, and Ureaplasma which were translated with codon table 4 according to their taxonomy using the additional orfipy argument “--table 4.” Kofam_scan tool was run on each orfipy output using the curated KO list of MEP and MVA genes of interest and pHMM profiles from the KOfam database. The kofam_scan command used was “./exec_annotation --format = detail-tsv -o ~ path_to_output/$output_file_name ~path_to_input/$input_file_name.”For each genome, orfipy was used to identify ORFs of at least 150 nucleotides in length and generate an output of the peptide translation using the specifications “--min 150 --pep $file_name.fa” . All gen2.3.For Dxs, DxrI, IspE, IspDF, IspG, IspH, HMGS, HMGRI, HMGRII, MVK, DPMD, Rubisco-like protein MTRu-1P isomerase, and cupin type I MTXu-5P methylsulferase a gene ortholog was determined to be present if the similarity score from alignment exceeded the KOfam score threshold for identifying similarity . This ta−10. The KOfam database did not have an adaptive threshold cutoff for DxrII for determination of orthology. For DxrII, we set the score cutoff at alignments with e-values of less than 1−10 as well.The computation of the KOfam score thresholds is described in detail in To determine a species’ MEP and MVA gene status, we sorted for presence and absence using Excel. Full table output of gene presence or absence is available in 2.4.Diversity exists in the lower part of the MVA pathway  but comp2.5.Species trees were constructed using Genome Taxonomy Database (GTDB) master tree  and onlyFor protein trees, the protein sequences corresponding to the entries from the species tree were extracted and aligned by MAFFT  using de3.3.1.To find candidate species for a non-canonical MEP pathway, we started with an available database of representative bacterial genomes  and extrMycoplasma, Rickettsia, and Spiroplasma  had all 7 MEP genes detected  but lack the MVA pathway according to our criteria (see Methods) . Some sproplasma . The remroplasma .Rhodospirillum rubrum   and thusm rubrum . The MTAm rubrum . For them rubrum . In thesFor the remaining 172 candidate species (95%) missing Dxs, the specific mechanism that allows the absence of Dxs requires further investigation to determine. Regardless of the mechanism, the identification of candidate species that contain all other MEP enzymes except Dxs suggest that the MEP pathway has evolutionary flexibility for the enzymatic activity that Dxs provides.3.2.Dxs is the only MEP pathway enzyme for which we identified a subset of candidate species that lack Dxs suggesting that an alternative strategy that circumvents Dxs has evolved. For IspG and IspH, there were no promising candidates for identification of alternative MEP pathways that circumvent one or both enzymes . Thus, wLelliotia steviae  used the MVA pathway and possess one or two MEP pathway enzymes whereas the remaining 8 species (23%) encode complete MEP pathways but no MVA pathway enzymes (Cellulomonas, 9 of the species encode a complete MEP pathway with no MVA enzymes but a single species, Cellulomonas taurus, encodes an incomplete MEP pathway and a complete MVA pathway (Cellulomonas taurus are in two operons in the genome, roughly 1 million base pairs apart.For example,  enzymes  (Misic e enzymes . In Cell pathway . The MVAThese data suggest that selective pressure exists to retain a terpenoid biosynthesis strategy, but that a species may use MEP, MVA, or both based on evolutionary trajectory.3.5.To understand the impact of horizontal gene transfer on the MEP pathway in bacteria, we generated evolutionary trees from protein sequences for Dxs, DxrI, IspE, IspG, and IspH . The DxsIspG is generally a two-domain protein in Bacteria, but a third domain called A* may be present . The A* Based on the differences between the MEP protein trees and the species tree, MEP pathway inheritance is not strictly vertical. Therefore, we suggest that horizontal gene transfer may have played a role in the evolution of this metabolic pathway.4.In this study, we assessed the MEP and MVA pathways and protein diversity across bacteria. Our goal was to inform options for engineering the precursor pathways for microbial terpenoid biomanufacturing by determining if alternatives exist for some steps in precursor pathways. In particular, we were interested in alternatives to Dxs and whether non-canonical MEP pathways can circumvent the IspG and IspH enzymes, which are known to pose major engineering challenges. To achieve this goal, we identified genomes lacking the MVA pathway, with an incomplete MEP pathway referred to as candidate species for a non-canonical MEP pathway. We found that Dxs is evolutionarily flexible and that alternatives may exist whereas for IspG and IspH we could not identify any candidate species that appear to circumvent these enzymes. These results may aid the ongoing quest to understand early evolution of terpenoid biosynthetic pathways.Identified alternatives to the canonical MEP enzymes include DxrII, the MTA-isoprenoid shunt, and mutations that arise when Dxs is knocked out . The supWe identified 181 candidate species for a non-canonical MEP pathway missing Dxs. In contrast, we identified fewer than 40 species missing IspG, IspH, or both in combination with other MEP enzymes and no compelling patterns among these species . We concTaken together, our results suggest that within the MEP pathway, evolutionary flexibility exists for Dxs. Alternative strategies to circumvent Dxs have likely evolved whereas IspG and IspH are evolutionarily rigid and no alternatives appear to have evolved or can be identified at this time. DxrI/II, IspD, IspE and IspF are also evolutionarily rigid as they appear to be retained by essentially all species using the MEP pathway. Whether the evolutionary rigidity of IspG, IspH, DxrI/II, IspD, IspE and IspF is due to a functional constraint hindering biochemical ability or due to the lack of sequence diversity available for the enzyme to explore under an evolving landscape remains an open question.The evolutionary rigidity of enzymes in the MEP pathway may be valuable for unraveling the evolution of terpenoid biosynthesis. Analysis of the protein phylogenetic trees for the MEP enzymes was consistent with the possibility that horizontal gene transfer has played a role in the evolution of these enzymes . The rolSpecies retaining all MEP enzymes except Dxs, are distributed across different phylogenetic groups . It is pA limitation of our bioinformatic approach is that it will only identify alternative MEP pathways in species lacking redundancy. Species with a complete MEP or MVA pathway in addition to an alternative non-canonical MEP pathway would not be identified. Additionally, we only surveyed the current database of bacterial species, which is inherently biased toward culturable bacteria and does not capture the full diversity of life . It is pOur findings also inform metabolic engineering of the MEP pathway in two ways. First regarding Dxs, we find that alternative enzymes to Dxs may exist. As Dxs governs MEP pathway flux via feedback regulation  and requThe development of sequencing technologies has enabled the determination of unprecedented numbers of bacterial genomes. This database of bacterial genomes is a repository of knowledge with astounding potential. Here, we leveraged the current repository of bacterial genomes as a resource for identifying alternative enzymes to enable engineering of a key metabolic pathway. Future efforts in microbial engineering should also utilize the database of bacterial genomes to search for naturally evolved alternatives for cellular metabolism that go beyond currently characterized literature.The original contributions presented in the study are included in the article/BM: Conceptualization, Data curation, Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. KA: Software, Visualization, Writing – review & editing. MW: Software, Supervision, Writing – review & editing. BK: Conceptualization, Funding acquisition, Supervision, Writing – review & editing. RL: Conceptualization, Funding acquisition, Supervision, Writing – review & editing.


Bovine viral diarrhea virus (BVDV) is a viral pathogen that poses a significant threat to the global cattle industry. BVDV infection interferes with the host’s innate and adaptive immunity, thereby affecting the diverse organs and systems of the body. Thus, comprehending the biological mechanisms by which viruses infect hosts is pivotal for disease control and prevention. Currently, the molecular mechanisms of the cell signaling pathways affected by BVDV infection remain unclear. Transcriptomic and proteomic methods offer promising avenues to dissect the biological processes in virus-infected host cells. In this study, transcriptomics and proteomics techniques were used to analyze differentially expressed proteins and scrutinize differentially expressed genes in immune-related biological processes after BVDV-1 infection in cow peripheral blood mononuclear cells (PBMCs). Our findings indicate that BVDV-1 predominantly suppresses the host through pathways such as the interleukin-17 signaling pathway, cytokine-cytokine receptor interaction, and complement and coagulation cascades. Conversely, PBMCs seem to counter viral invasion through mechanisms such as complement and coagulation cascades, the RIG-I-like receptor signaling pathway, and cytokine-cytokine receptor interaction. This study forms a theoretical basis for analyzing the mechanism of BVDV-1 infection and facilitates a deeper exploration of host responses to BVDV-1 infection in dairy cows.Bovine viral diarrhea virus (BVDV) causes bovine viral diarrhea-mucosal disease, inflicting substantial economic losses upon the global cattle industry. Peripheral blood mononuclear cells (PBMCs) are the central hub for immune responses during host-virus infection and have been recognized as crucial targets for BVDV infection. In order to elucidate the dynamics of host-BVDV-1 interaction, this study harnessed RNA-seq and iTRAQ methods to acquire an extensive dataset of transcriptomics and proteomics data from samples of BVDV-1-infected PBMCs at the 12-h post-infection mark. When compared to mock-infected PBMCs, we identified 344 differentially expressed genes  and 446 differentially expressed proteins . Selected DEGs and DEPs were validated through quantitative reverse transcriptase-polymerase chain reaction and parallel reaction monitoring. Gene ontology annotation and KEGG enrichment analysis underscored the significant enrichment of DEGs and DEPs in various immunity-related signaling pathways, including antigen processing and presentation, complement and coagulation cascades, cytokine-cytokine receptor interaction, and the NOD-like receptor signaling pathway, among others. Further analysis unveiled that those DEGs and DEPs with downregulated expression were predominantly associated with pathways such as complement and coagulation cascades, the interleukin-17 signaling pathway, cytokine-cytokine receptor interaction, the PI3K-Akt signaling pathway, the tumor necrosis factor signaling pathway, and the NOD-like receptor signaling pathway. Conversely, upregulated DEGs and DEPs were chiefly linked to metabolic pathways, oxidative phosphorylation, complement and coagulation cascades, and the RIG-I-like receptor signaling pathway. These altered genes and proteins shed light on the intense host-virus conflict within the immune realm. Our transcriptomics and proteomics data constitute a significant foundation for delving further into the interaction mechanism between BVDV and its host. Pestivirus in the family Flaviviridae , pH 10) ranging from 2% to 90% was utilized over 60 min, followed by high-pH separation (maintaining a flow rate of 0.3 mL/min and a temperature of 30 °C). Individual components were collected and subsequently vacuum-dried. For each sample, a 2-μL total peptides sample was subjected to separation and analyzed by a nano-UPLC (EASY-nLC1200) coupled to a Q Exactive HFX Orbitrap instrument (Thermo Fisher Scientific) equipped with a nano-electrospray ion source. To conduct the experiment, we utilized a reversed-phase column  measuring 100 μm ID × 15 cm. The column allowed for efficient separation of our samples. The mobile phases employed in the separation process consisted of two solutions: H2O containing 0.1% FA and 2% ACN (phase A) and phase B consisting of 80% ACN and 0.1% FA. To achieve separation, a gradient lasting 90 min was applied at a flow rate of 300 nL/min. The gradient was designed as follows: B: 2–5% (2 min), 5–22% (68 min), 22–45% (16 min), 45–95% (2 min), and 95% (2 min). The execution of data-dependent acquisition (DDA) followed a profile and positive mode approach, employing an Orbitrap analyzer. The resolution for MS1 was set at 120,000 (@200 m/z), and the m/z range was specified as 350–1600. In the case of MS2, a resolution of 45,000 was selected while maintaining a fixed first mass of 110 m/z. To ensure optimal control, the automatic gain control (AGC) target for MS1 was established at 3×106 with a maximum integration time (IT) of 30 ms. Similarly, for MS2, the AGC target was set to 1E5 with a maximum IT of 96 ms. Fragmentation of the top 20 most intense ions occurred through higher-energy collisional dissociation (HCD) technique with a normalized collision energy (NCE) of 32%. The isolation window for HCD was determined to be 0.7 m/z. Additionally, a dynamic exclusion time window of 45 s was implemented, encompassing both single-charged peaks and peaks exceeding a charge of six, thereby excluding them from the DDA process.After vacuum drying, the polypeptides were re-dissolved in buffer A  at 50 °C. Subsequently, separation was carried out using RPUPLC  under alkaline conditions. A linear gradient of buffer B . Subsequently, the DEPs underwent GO database analysis and KEGG database analysis to annotate their functional properties and enrich their associated pathways, respectively. All original sequence reads were submitted to the NCBI database as a BioProject (PRJNA1003857).The mass spectrometry data were employed by Proteome Discoverer (PD) software (Version 2.4.0.305) and its integrated Sequest HT search engine. The UniProt FASTA databases at the species level (uniprot-Bos + taurus (Bovine)-9913-2020-10) were employed to conduct searches of the MS spectra lists. Both the PSM and peptide levels had the false discovery rate (FDR) set at 0.01. Peptide identification involved a deviation of up to 10 ppm for the initial precursor mass and 0.02 Da for the fragment mass. Protein quantification utilized a unique peptide and Razor peptide, while the total peptide amount was used for normalization. Quantitative analysis was conducted on proteins with at least 2 unique spectra, with a significance threshold of m/z. Subsequently, MS/MS fragmentation was achieved through higher-energy collisional dissociation (HCD) utilizing an NCE value of 35. Additional settings for MS/MS included a resolution of 15,000 and an AGC target value of 5×104. The maximum ion injection time was once again set to 50 ms, while dynamic exclusion was established at 15.0 s to prevent repeated fragment analysis. In order to perform PRM mode testing, the parameter “chromatography in the DDA PaSEF mode” was selected. To integrate the desired peptide lists obtained from DDA results into the inclusion list of the Xcalibur PRM method editing module, Skyline software was employed. The following PRM parameters were configured: an MS2 resolution of 15,000, an isolation window of 1.6 m/z, an AGC value of 5×104, a maximum accumulated time of 60 ms, and an HCD energy of 35. Both the control and test groups were thoroughly evaluated using four independent biological replicates. Fourteen DEPs were randomly selected for targeted quantification by parallel reaction monitoring (PRM) conducted by Biotree Biotech Co., Ltd., . The peptides utilized for PRM analysis were synthesized following the established protocol used for TMT. To conduct a DDA mode test, the PRM samples were loaded onto a trap column  at a flow rate of 5 μL/min for a duration of 10 min. Next, each individual sample underwent separation on a C18 reversed-phase column  using a linear gradient. For optimal mass resolution, the primary setting was adjusted to 60,000, with the AGC target value being set at 4e5. The maximum ion injection time was defined as 50 ms. Full MS scans were acquired within the mass range of 100–1700 p < 0.05 or q < 0.05. Statistical analysis was performed using SPSS v.22.0 software .All data were presented as mean with standard error of the mean. Comparisons between the two groups were performed using Student’s t-test. Differences were considered statistically significant when In order to assess the growth characteristics of BVDV on PBMCs, we utilized RT-PCR and qRT-PCR techniques to validate the replication of BVDV-1 in cow PBMCs. As depicted in ™ 6000 platform. After RNA sequencing, we obtained 323,896,256 raw reads . After removing the low-quality reads and reads with adapter sequences, 314,906,984 valid reads were obtained ”. Regarding the NC and V samples, the reference genome obtained 156,345,812 and 141,183,185 mapped reads, respectively. The corresponding mapping rates for both samples were 94.54% and 94.41% . This validates the reliability of the DEGs identified through RNA-Seq. Similarly, the PRM analysis of DEPs confirmed the abundance levels of 14 DEPs, which aligned with the iTRAQ proteomics analysis . This recognition process triggers pathways such as the Toll-like signaling pathway and the RIG-I signaling pathway, resulting in a surge of antiviral cytokines ,31. PBMCUpon pathogen invasion, the innate immune response is the first line of action. Inflammatory cytokines play a pivotal role in activating innate immune cells, initiating complement activation, and triggering specific immune responses. Inflammatory cytokines such as IFN, IL, and tumor necrosis factor (TNF) activate specific signal transduction pathways to realize their biological functions. Our study revealed that the DEGs altered during BVDV-1 infection of PBMCs were prominently enriched in the cytokine-cytokine receptor interaction KEGG pathway. Of the 11 DEGs involved in this pathway, seven genes  showed significantly downregulated expression, whereas four genes showed significantly upregulated expression . These findings align with studies conducted on BVDV-infected MDBK cells, where DEGs were primarily related to cytokine-cytokine receptor interaction, with the downregulated genes associated with immune processes, cell death, and stimuli . SimilarThe complement system, comprising over 35 soluble plasma proteins, constitutes a crucial element of innate defense, bridging innate and adaptive immune responses and actively preventing infections ,56. Our In summary, the analysis of RNA-Seq and iTRAQ data revealed a series of immune-related DEGs and DEPs in the peripheral blood of BVDV-1-infected dairy cows. The study’s findings underscore that BVDV-1 primarily suppresses and evades the host through pathways like the IL-17 signaling pathway, cytokine-cytokine receptor interaction, the PI3K-Akt signaling pathway, complement and coagulation cascades, the TNF signaling pathway, and the NOD-like receptor signaling pathway, ultimately achieving PI. Concurrently, the host seems to activate innate immunity against viral invasion through pathways such as complement and coagulation cascades, the RIG-I-like receptor signaling pathway, and cytokine-cytokine receptor interaction. These results deepen our understanding of BVDV-host interactions during BVDV-1 infection and offer novel insights for comprehending the intricate dynamics of host-virus survival mechanisms.


Diabetic Nephropathy (DN) is a serious complication of diabetes, the diagnosis and treatment of DN is still limited. Sinomenine (SIN) is an active extract of herbal medicine and has been applied into the therapy of DN.In the part of bioinformatic analyses, network pharmacology and molecular docking analyses were conducted to predict the important pathway of SIN treatment for DN. In-vivo study, DN rats were randomized to be treated with vehicle or SIN (20 mg/kg or 40 mg/kg) daily by gavage for 8 weeks. Then, the pharmacological effect of SIN on DN and the potential mechanisms were also evaluated by 24 h albuminuria, histopathological examination, transcriptomics, and metabolomics.Firstly, network pharmacology and molecular docking were performed to show that SIN might improve DN via AGEs/RAGE, IL-17, JAK, TNF pathways. Urine biochemical parameters showed that SIN treatment could significantly reduce 24 h albuminuria of DN rats. Transcriptomics analysis found SIN could affect DN progression via inflammation and EMT pathways. Metabolic pathway analysis found SIN would mainly involve in arginine biosynthesis, linoleic acid metabolism, arachidonic acid metabolism, and glycerophospholipid metabolism to affect DN development.We confirmed that SIN could inhibit the progression of DN via affecting multiple genes and metabolites related pathways. Diabetic Nephropathy (DN) refers to kidney damage caused by diabetes, which is a common and serious complication of diabetes leading to the development of end-stage kidney disease . EpidemiFor decades, there is nearly no effective treatment for DN though the pathological mechanisms of DN have been deeply studied , and DN Network pharmacology is becoming a common methodology to predict the potential targets of a drug or compound to treat the disease . In addiStreptozotocin (STZ) was purchased from Sigma-Aldrich. Sinomenine (purity > 98%) was purchased from Shanghai Roche Pharmaceutical Co., Ltd. . Urinary protein concentration was estimated with a protein assay kit . TRIzol® Reagent was purchased from Invitrogen. Hematoxylin, eosin and periodic acid-Schiff were from Wuhan servicebio technology CO., Ltd. .https://www.genecards.org/), a database with data about genomics, proteomics, and transcriptomics oxidanesulfonic acid; (2 S)-2-amino-3-(4-hydroxyphenyl)propanoic acidL-Tyrosine; Naringenin-7-O-beta-D-Glucuronide; Furanogermenone; 3,5-dichlorosalicylic acid; Rac-5,6-Epoxy-retinoyl-beta-D-glucuronide; 3,4,5-trihydroxy-6-[(2-pentyl-3-phenyloxiran-2-yl)methoxy]oxane-2-carboxylic acid; Ganglioside GT3 (d18:1/20:0); N2-Acetyl-L-ornithine, which were showed in Table To obtain a comprehensive view of the mechanisms of SIN against DN, we constructed an interaction network based on metabolomics and transcriptomics. The key metabolites and putative genes in DN versus SIN (20 mg/kg) and SIN (40 mg/kg)-treated group were loaded into MetScape (a plugin in of Cytoscape), to establish a metabolite–reaction–enzyme–gene network in order to visualize the molecular mechanism of SIN in treating DN. In DN versus SIN (20 mg/kg) group, we found S--glutathione was significantly upregulated and 9-deoxy-delta12-PGD2 was significantly downregulated in prostaglandin formation from arachidonate. Deoxycytidine was significantly upregulated in pyrimidine metabolism. In addition, N-Acetylornithine, L-Proline and the expression of GPT2 were significantly downregulated in urea cycle and metabolism of arginine, proline, glutamate, aspartate and asparagine. And Rac-5,6-Epoxy-retinoyl-beta-D-glucuronide was significantly increased in vitamin A (retinol) metabolism Fig. .In DN versus SIN (40 mg/kg) group, the expression level of PLA2G2A was significantly increased in arachidonic acid metabolism, while PTGS2 and CYP1A1 were significantly downregulated. PLA2G2A was upregulated and compound L-Serine was downregulated in Glycerophospholipid metabolism. In leukotriene metabolism, we found the expression of ACSL6 was significantly increased and CYP1A1 was decreased. In linoleate metabolism, PLA2G2A was significantly upregulated and PTGS2 was downregulated. In purine metabolism, the expression of ATP12A and ADA were significantly increased while the expression of POLR2H and PDE10A were significantly decreased. In addition, ASMT and CYP1A1 were significantly downregulated in tryptophan metabolism. In tyrosine metabolism, CRYZ was significantly upregulated and CYP1A1 was downregulated. Compound L-Citrulline and L-Asparagine were significantly upregulated and N-Acetylornithine was downregulated in urea cycle and metabolism of arginine, proline, glutamate, aspartate and asparagine. The expression of CYP1A1 was downregulated in xenobiotics metabolism Fig. .DN is a serious microvascular complication of diabetes and is the leading cause of ESRD . SinomenOur transcriptomics results indicated that DEGs involved multiple classical biological processes, including phosphatidylinositol 3-kinase regulator activity, Hsp90 protein binding, very long-chain fatty acid metabolic process, hyaluronic acid binding, positive regulation of collagen metabolic process, positive regulation of collagen biosynthetic process. These biological processes contribute to the processes of renal fibrosis , disturbCombined the results of network pharmacology and transcriptomics, we found JAK-STAT signaling pathway is a most important pathway which may be involved in the mechanisms of SIN treating DN. The JAK-STAT signaling involved in key mechanisms for several cytokines and growth factors . JAK canOur metabonomics results showed that SIN might improve DN via several metabolites and some metabolites have been found to be associated with the development of DN. Arbutin has anti-oxidative and anti-inflammatory activities, which could markedly improve renal function, and attenuate inflammation and cell apoptosis by modulating PI3K/Akt/Nrf2 signalling in acute kidney injury . PhenilaUric acid is an end product of the purine metabolism and excreted predominantly by the proximal tubules. For patients with DN, higher uric acid levels are associated with higher microalbuminuria, lower eGFR ; LinoleiIn our study, we firstly used a novel combined strategy to identify the potential targets and mechanisms of SIN administration in treating DN according to metabolomics, transcriptomics, histology, biochemical parameters and network pharmacology. It will provide a new paradigm to figure out the potential mechanisms of pharmacological effects of a natural compound. In addition, our research offered information and theoretical foundation for an in-depth observation of mechanisms and provided the support for SIN clinical practice. Future systematic molecular biology experiments will be needed to confirm the precise mechanism.


Organophosphate pesticides (OPs) are toxic substances that contaminate aquatic environments, interfere with the development of the nervous system, and induce Neurodevelopmental Toxicity (NDT) in animals and humans. The canonical mechanism of OP neurotoxicity involves the inhibition of acetylcholinesterase (AChE), but other mechanisms non-AChE are also involved and not fully understood. We used network toxicology and molecular docking to identify molecular targets and toxicity mechanisms common to OPs. Targets related to diazinon-oxon, chlorpyrifos oxon, and paraoxon OPs were predicted using the Swiss Target Prediction and PharmMapper databases. Targets related to NDT were compiled from GeneCards and OMIM databases. In order to construct the protein–protein interaction (PPI) network, the common targets between OPs and NDT were imported into the STRING. Network topological analyses identified EGFR, MET, HSP90AA1, and SRC as hub nodes common to the three OPs. Using the Reactome pathway and gene ontology, we found that signal transduction, axon guidance, cellular responses to stress, and glutamatergic signaling activation play key roles in OP-induced NDT. The widespread use of organophosphate pesticides (OPs) in agriculture still raises concerns about their ecological impact and human health implication . SurfaceThere are numerous toxic effects associated with OPs in fish, including hematological disturbances, gill, kidney, and liver alterations , oxidatiExposure to OPs poses a high risk of neurotoxicity, especially for the developing nervous system. The effects of this type of exposure can include cognitive, motor, and memory impairments, as well as changes in brain morphology, referred to as Neurodevelopmental Toxicity (NDT) .Acute exposure to high doses of OPs, such as self-poisoning, can significantly inhibit the activity of the acetylcholinesterase (AChE) enzyme. As a result, acetylcholine accumulates in the synaptic cleft, and cholinergic receptors in the nervous system and neuromuscular junction are overactivated, leading to a cholinergic crisis. The symptoms of human poisoning include respiratory depression, cardiovascular complications, gastrointestinal problems, and mental confusion, and the mortality rate is 15–30% .2+ homeostasis, neuronal activity [When environmental contamination occurs, OPs concentrations are typically low and may not significantly inhibit AChE activity. However, these low concentrations can still cause behavioral alterations and neurological damage, particularly during developmental stages . Therefoactivity , the modactivity .Even though AChE inhibition is a common mechanism of action for OPs, there is evidence that the non-AChE effects can vary among different OP types. For instance, chlorpyrifos (CP) and malathion exhibited contrasting effects on zebrafish larvae. While malathion treatment led to morphological abnormalities in the brains of larvae, no such effect was observed with CP. Furthermore, CP-exposed larvae exhibited decreased swimming speed and increased resting time, whereas malathion-exposed larvae showed increased swimming speed and decreased resting time .A comparative analysis of seven OPs  in planarians revealed shared toxicological endpoints among the OPs (except for acephate and parathion), such as abnormal body forms, increased viscosity, writhing behavior, and alterations in swimming speed during exposure to blue light. However, other toxicological endpoints differed between the OPs .This study was motivated by the following question: Do OPs share non-AChE mechanisms that contribute to NDT? This information can be valuable in enhancing toxicity testing protocols, establishing more precise safety margins, and providing targets for more effective treatments in cases of exposure to these compounds.Computational tools can enhance toxicity studies by providing fast and comprehensive results. Network toxicology, a branch of network pharmacology, is one of these tools.Network pharmacology, an emerging approach in the pharmaceutical industry, allows the analysis of molecular and protein interactions, as well as the mechanisms of action, prediction of therapeutic targets and signaling pathways in drug development . HoweverCombining network toxicology and molecular docking can reveal interactions between the investigated compounds and their predicted targets, thereby helping to determine their toxicological properties . Thus, nThe aim of this study was to predict non-AChE targets that are shared among OP pesticides that contribute to NDT using network toxicology and molecular docking. https://pubchem.ncbi.nlm.nih.gov) accessed on 28 February 2023. We chose the oxon metabolites over of the original compounds due to their direct association with NDT [The 2D structures of the oxon metabolites of the OP pesticides diazinon (DZ), CP, and parathion were retrieved from the PubChem database (with NDT . The imawith NDT  and are http://www.swisstargetprediction.ch) accessed on 28 February 2023 and PharmMapper (http://www.lilab-ecust.cn/pharmmapper/) accessed on 28 February 2023 to predict target genes. We then entered the predicted target genes into the UniProt database (https://www.uniprot.org/) accessed on 28 February 2023 to retrieve the standard gene names, and duplicate entries were eliminated.The 2D chemical structures of paraoxon (PO), chlorpyrifos oxon (CPO), and diazinon-oxon (DO) were submitted to the Swiss Target Prediction database (https://www.genecards.org/) accessed on 28 February 2023 and Online Mendelian Inheritance in Man  accessed on 28 February 2023 were used to predict potential NDT targets. In February 2023, the related targets were collected using the keywords “neurodevelopmental abnormalities”, “neurotoxicity”, and “neurodevelopmental disorders”, along with the Homo sapiens species. The UniProt database was utilized to retrieve the standard gene names, and after merging the predicted targets for the three keywords from both databases, duplicate entries were removed.The GeneCards database (http://bioinformatics.psb.ugent.be/webtools/Venn/) accessed on 28 February 2023.In order to identify the intersection genes as potential targets of each OP related to NDT, a Venn diagram was created by using Draw Venn Diagram tool (https://string-db.org/) accessed on 28 February 2023. A minimum score of 0.7 was set, indicating a high level of confidence, and only experimental data were selected as the source of active interaction. The PPI network analyzes direct interactions between the inputted proteins and generates a network of interactions where proteins are represented as nodes and the interactions between them are represented as edges.Protein–protein interaction (PPI) networks were constructed using the STRING database  method. The Analyze Network tool was used for topological analyses of the network, including degree (k), betweenness centrality (BC), clustering coefficient, closeness centrality (CC), and average shortest pathway (ASPL).The measure k represents the number of connections a node has with other nodes in the network, i.e., it quantifies the number of edges that connect to it. Nodes with a high k value are important for information transmission in the network as they have more control over the flow of information. On the other hand, BC is a measure that indicates a node’s ability to connect other nodes in the network. A high BC value indicates that the node is important for communication between different parts of the network. In turn, the CC measure indicates how many direct neighbors of a node are interconnected, and a high CC value for a node indicates its involvement in a strongly connected cluster. Lastly, the ASPL measure indicates the average shortest path length between a node and all other nodes in the network .http://cpdb.molgen.mpg.de/) accessed on 28 February 2023 to explore the Gene Ontology (GO) and Reactome Pathway. The Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) categories of GO were chosen. GO terms and Reactome Pathways with a p-value < 0.01 were considered statistically significant. The top 15 GO terms for BP, MF, and CC, as well as the top 30 Reactome pathways, were selected for each OP. Additionally, terms common to all three OPs that were directly related to the Nervous System superpathway, as classified by Reactome.org, were examined. GO and Reactome pathway graphs were generated with the ggplot2 package in RStudio.An enrichment analysis was performed using the Consensus PathDB database ((a)https://www.rcsb.org/) accessed on 28 February 2023 in PDB format. Discovery Studio 2021 was used to remove water molecules and ligands, add polar hydrogen bonds, and obtain the x, y, z coordinates for constructing the grid box. Energy minimization was performed using Swiss-PDB Viewer 4.1.0 software.Macromolecule preparation: The 3D structures of the proteins were obtained from the PDB database ((b)https://pubchem.ncbi.nlm.nih.gov/) accessed on 28 February 2023 in SDF format and converted to PDB format using Open Babel.Ligand preparation: The 3D structures of the ligands were retrieved from the PubChem database ((c)Docking simulation was performed using Autodock Vina , using a(d)The analysis of the binding and generation of images was carried out using Discovery Studio 2021. The distance criterion between ligands and amino acid residues was established as <3.3 Å for hydrogen bonds  and <6.0A molecular docking analysis was conducted to explore the potential interactions between OPs and their hub nodes. The analysis followed the following steps:After removing the duplicates, 388 targets were found for DZO, 343 for CPO, and 387 for PO in the Swiss Target Prediction and PharmMapper databases. Additionally, 3060 NDT-related targets were obtained from the Gene Cards and OMIM databases after removing the duplicates. Using a Venn diagram to identify the intersections of targets for each OP and NDT, we found 187, 121, and 181 targets for DZO, CPO, and PO, respectively. These targets were used to construct a PPI network using the STRING database, and the data were imported into Cytoscape for visualization and analysis of the hub nodes.The analysis of PPI networks revealed 81 nodes and 113 edges for the DZO-NDT network, 37 nodes and 50 edges for the CPO-NDT network, and 71 nodes and 103 edges for the PO-NDT network . The topp-value < 0.01). We found 169 GO terms for DZO, with 102 in BP, 20 in MF, and 47 in CC. For CPO, we identified 143 GO terms, with 89 in BP, 18 in MF, and 36 in CC. PO had 155 GO terms, with 98 in BP, 18 in MF, and 39 in CC. For each OP, we selected the Top 15 processes for each term based on their p-values . p-value.Based on Reactome pathway analysis, 419 pathways were identified for DZO, 254 for CPO, and 358 for PO  and their subdivisions . Accordip-value < 0.01) and were classified under the Neuronal system superpathway to identify the key processes in the nervous system affected by OP exposure. Activation of NMDA receptors and postsynaptic events was the common pathway for all three OPs, indicating the activation of N-methyl-D-aspartate receptors (NMDAR), an ionotropic glutamate receptor.We also evaluated the Reactome pathways for DZO, CPO and PO that showed significant values  generation and oxidative stress, which may contribute to mitochondrial damage and cell death ,37,38. IBased on our literature review, no experimental data were found, indicating that OPs cause neurodevelopmental abnormalities via EGFR, MET, or non-receptor tyrosine kinase protein SRC. However, CP has previously been shown to increase migration and invasion of breast cancer cells through c-SRC pathway activation, amplifying downstream AKT and p-38 signaling . AdditioAxon guidance is the process by which neurons send their axons toward their correct targets for synaptic formation, and it is crucial during neural circuit development. Growing axons have a structure at their tips known as the growth cone, which contains receptors that respond to extracellular messengers that attract or repel the axon, guiding them to their destination . The hubThe EGFR (or ErbB1) belongs to the ErbB family, which includes three other members: ErbB2, ErbB3, and ErbB4. Activation of EGFR initiates signaling pathways that are important for neuronal proliferation, differentiation, migration, as well as neural circuit development, including the Ras-Raf-MEK-ERK1/2, STAT3, and PI3K-Akt-mTOR pathways . AdditioAxonal growth is a fundamental process in the formation of neural circuits and the development of neuronal connectivity. Previous studies have reported impairment of axonal growth by OPs through noncholinergic mechanisms. For example, low concentrations of CP and CPO inhibited axonal outgrowth in cultures of rat sympathetic neurons . MoreoveHSP90AA1 gene encodes the Heat shock protein (Hsps) HSP90, which acts as a chaperone to refold denatured proteins in the presence of stressors such as heat, oxidative stress, hypoxia, and exposure to cytotoxic agents. In the absence of stress, chaperones facilitate protein refolding, stabilization, translocation, and degradation, preventing protein aggregation. In neurodevelopment, Hsps regulate pathways related to cell growth and migration, such as the PI3K/Akt signaling pathway, and they are crucial mediators for axon guidance [Exposure to environmental contaminants during prenatal development can trigger cellular response to stress that is closely related to heat shock and oxidative stress systems . The HSPguidance .Cellular stress increases the expression of HSP90 and diverts its functions to cope with stress responses. This functional diversion or inhibition of HSP90 can alter the activity of various proteins and result in increased mutations, leading to neurodevelopmental disorders . The incThere are several ways cells respond to stress, including activating of survival pathways or apoptosis . There iThe ionotropic glutamate receptors N-Methyl-D-Aspartate receptors (NMDAR) are calcium-permeable channels. The synaptic activity of NMDAR contributes to axonal and dendritic growth, as well as the maturation of glutamatergic synapses . These pDuring exposure to high concentrations of OPs, ionotropic AMPA and NMDA glutamate receptors are activated due to increased excitatory signaling following AChE inhibition and excessive stimulation of muscarinic receptors, leading to seizures and neuronal death . HoweverStudies indicate that glutamate-mediated excitotoxicity seems to be more related to the toxicity effects of CP than DZ. An NMDAR antagonist, MK-801, attenuated CP toxicity, but not DZ toxicity, in cortical neuronal and glial cultures . FurtherIn our study, we did not assess the direct activation of NMDAR by OPs, nor the genes encoding the subunits of this receptor. However, the activity of NMDAR can be regulated by SRC , EGFR 6, and METUsing network toxicology and molecular docking analyses, this study identified the hub nodes HSP90AA1, EGFR, MET, and SRC as potential targets for OPs. Additionally, signal transduction, axon guidance, cellular response to stress, and the activation of NMDAR were found to be key pathways involved in OP-induced NDT. These findings are important for understanding the mechanisms of neurotoxicity of these substances at environmentally relevant concentrations that do not involve cholinergic pathways.