Strychnos nux-vomica L.  against papillary carcinoma thyroid (PTC) by combined of network pharmacology and experimental verification. By searching the TCMSP, SEA and SwissTarget Prediction database, the main active ingredients and related targets were obtained. Utilizing Venny 2.1.0 String database and Cytoscape 3.7.2 to screened the intersection target and constructed protein–protein interaction (PPI) network diagram. Using R 4.0.4 software carried out the enrichment analysis of GO and KEGG. HPLC was carried out using LC-20A modular HPLC system to identify the bioactive compound brucine present in SS. Molecular docking was performed using Discovery 2019 software. The inhibition rate was detected by CCK8 method. Western blot was used to detect the expression levels of brucine anti-PTC related pathway proteins. 14 active components were screened out, of which 4 main components showed tight relationship with PTC. SS may play the anti-PTC role by acting on two main pathways  and mediating various biological functions. HPLC analysis revealed that brucine was a suitable marker for standardization of the SS. 4 active components exhibit strong binding energy with core protein. Brucine could significantly reduce the activity of BCPAP cells compared with isobrucine, stigmasterol, (+)-catechin. Brucine may reduce the protein expression levels of IL-6, VEGFA, JUN, TP53, 1L1B, PTGS2, BCL2, CASP3, CASP8, and CASP9 while increase the protein expression levels of BAD, cleaved-CASP3, cleaved-CASP8, and cleaved-CASP9 in BCPAP cells, respectively. The active components of SS against PTC mainly include isobrucine, stigmasterol, (+)-catechin, brucine. Among them, brucine exhibits the strongest anti-PTC activity in BCPAP cells, which may reduce the PTC-related protein expression levels. Therefore, SS may exhibits the anti-PTC activities through multiple targets and pathways.The aim of the study was to investigated the mechanism of  In the past 30 years, the incidence rate of thyroid cancer in most regions of the world has continued to rise, and it has become the fastest growing solid tumor with incidence rate in the world2. According to the data of the Chinese National Cancer Registration Center (CNCHS), thyroid cancer in China will continue to grow at a rate of 4.5% every year. According to the final data which released by China Cancer Center (CCC) in 2017, the total incidence rate of thyroid cancer was about 10.16/105, ranking seventh in the incidence rate of malignant tumors4. Among them, PTC is the most common pathological type of thyroid cancer, accounting for more than 85% of thyroid cancer5. Most PTC patients have a good prognosis after traditional surgical treatment. Although the prognosis of most PTC patients is relative good, PTC will metastasize to the neck or distant lymph nodes in a few cases. The lymph nodes metastasized by PTC may invade important tissues such as blood vessels, nerves and trachea, resulting in increased difficulty in surgery, which may increase the risk of secondary or multiple operations for PTC patients, and finally affect the prognosis of patients7.Thyroid papillary carcinoma (PTC) is the most common type of differentiated thyroid carcinoma, accounting for 80–85% of the total incidence of thyroid malignant tumorsStrychnos nux-vomica L., , as the mature seed of Yunnan Strychnos nux or Strychnos nux, is a commonly used Traditional Chinese Medicine (TCM) in clinical practice. It belongs to the liver and spleen meridians, which exhibits the effects of dredging collaterals, relieving pain, dispersing knots and reducing swelling8. The chemical components of SS are complex and diverse, that mainly including alkaloids, phenolic acids, terpenoids, steroids and glycosides13. Modern pharmacological studies showed that SS has significant pharmacological effects in anti-tumor14, anti-rheumatism15, analgesia16, neuroprotection17 and other diseases. SS contains a variety of monomers and acts on a variety of cell targets. It is difficult to systematically study the synergistic mechanism of SS with conventional methods. Network pharmacology provides a new method for the complex molecular mechanism and the material basis of the efficacy of TCM18. Through the analysis of various complex and multi-level interaction networks, the synergistic effects of components and multi-objective drugs and their potential mechanisms are better described. It has been widely used to study the molecular mechanism of TCM, TCM pair and TCM compound on diseases19. Therefore, the present study focus on the anti-tumor targets of the active components of SS through network pharmacological methods, constructs a systematic visual network diagram for the components target diseases, and analyzes the Gene Ontology (GO) function enrichment and Kyoto Encylopaedia of Genes and Genomes (KEGG) signal pathway of target genes, thereby clarifying the potential molecular mechanism of SS on anti-PTC. At the same time, we verified the active substances of SS screened in the earlier stage at the cellular and protein levels, providing a basis for SS anti-PTC . Utilizing the oral bioavailability (OB) ≥ 30% and drug like (DL) ≥ 0.18 as screening conditions, all active components of SS were obtained. OB and DL are important indicators for screening active ingredients in different TCM respectively20. Using SEA database (http://sea.bkslab.org/) and SwissTarget Prediction databases (http://www.swisstargetprediction.ch/index.php) separately for target prediction of active ingredients of SS.All the chemical components are obtained by using https://www.genecards.org/), using “PTC”, “papillary thyroid microcarcinoma” and “papillary thyroid cancer” as keywords, search for PTC tumor related target genes, and set target genes with reliable evidence sources (Relevance score > 20). The UniProt database (http://www.uniprot.org/) is used to transform the targets and obtain the gene name of the corresponding targets.From Genecards database  to analysis the intersection target genes of active components related target genes and PTC related target genes, the common action target of disease and drug was obtained. Then using the Metascape database (http://metascape.org/), import the corresponding genes, and obtain Gene ID, the relevant genes of SS anti-PTC was finally acquired.Using R 4.0.4 softwareSS and the PTC-related target genes were matched to obtain the common genes. That is, the key targets of SS against PTC was obtained. Nodes in the network represent components, targets and diseases respectively; the edge in the network is used to connect drugs and active components, active components and target genes. The whole network showed the relationship among drugs, active components and targets. The “component-target-disease” network diagram was constructed by using Cytoscape 3.7.2 software22.The target genes corresponding to the active ingredients of https://cn.string-db.org/), select “Homes sapiens” as the species category, and obtain target interaction PPI network diagram. The results were exported in “TSV” format, and key target genes were screened by using Cytoscape 3.7.2 software.Input the intersection target genes obtained into the String online analysis platform (https://david-d.ncifcrf.gov/home.jsp) to carry out the GO function enrichment analysis and KEGG pathway enrichment analysis. After setting the threshold value to P < 0.05, take the top 20 GO term and KEGG channels, analyze them with clusterProfiler package of R 4.0.4 software23, and draw bubble chart and column chart respectively. In addition, to validated the anti-PTC mechanism of SS across the key targets and multiple pathways, the KEGG mapper functional analysis was used to mark the target genes on the pathway associated with PTC.Using DAVID database (SS was retrieved and downloaded respectively in PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Then the PDB file of core target protein structure was downloaded from RCSB database (https://www.rcsb.org/). The Discovery Studio 2019 software was used to verify the docking result between the screened compound small molecules and the core target protein macromolecule with the highest degree.The SDF files with 2D structure of monomer compounds from http://portal.gdc.cancer.gov/) and Genotype-Tissue Expression (GTEx) database . It provides customizable functions, such as tumor/normal differential expression analysis, clinical parameter analysis and survival analysis of tumor patients. Using the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database (http://gepia.cancer-pku.cn/) to further verify the expression results of IL-6, VEGFA, JUN, TP53, 1L1B and PTGS2. To analyze the differential expression of above genes in different stages of thyroid cancer and its relationship with survival and prognosis. Set the filter conditions in the “Stage Plot” module of expression DIY: (1) Gene: IL-6, VEGFA, JUN, TP53, 1L1B and PTGS2; (2) Use major stage: yes; (3) Data sets selection (cancer name): THCA; (4) Log Scale: yes. Set the filter conditions in the “survival plots” module: (1) Gene: IL-6, VEGFA, JUN, TP53, 1L1B and PTGS2; (2) Methods: Overall Survival (OS); (3) Group cutoff: median; (4) Hazards ratio (HR): yes; (5) 95% confidence interval: yes; (6) Axis units: months; (7) Data sets selection: THCA. GEPIA database is an online tool for analyzing tumor and normal samples. Its data source is The Cancer Genome Atlas (TCGA) database , stigmasterol (CAS No. 83-48-7), (+)-catechin (CAS No. 154-23-4), brucine (CAS No. 5892-11-5) were purchased from Selleck China company respectively. 1640 or DMEM culture medium, DMSO, fetal bovine serum, penicillin streptomycin double antibody, 0.25% trypsin, cell counting kit 8 (CCK-8), and DMSO purchased from Sangon Biotech (Shanghai) Co., Ltd.. RIPA lysis buffer, rabbit derived IL-6 antibody, JUN antibody, TP53 antibody, PTGS2 antibody, VEGFA antibody, 1L1B antibody, BAD antibody, BCL2 antibody, CASP3 antibody, CASP8 antibody, CASP9 antibody, cleaved CASP3 antibody, cleaved CASP8 antibody, cleaved CASP9 antibody, rabbit derived GAPDH/(beta-actin) antibody, HRP goat anti-mouse IgG(H + L) antibody and goat anti-rabbit IgG were purchased from Beyotime Biotechnology Co., Ltd., Nanjing Jiancheng Bioengineering Institute, Santa Cruz Biotechnology, and ThermoFisher Scientific Co., Ltd. respectively. The instrument includes BioRad full-automatic microplate reader, BioRad chemiluminescence gel imaging system, OLYMPUS IX51 inverted microscope, MCO-15AC SANYO CO2 constant temperature incubator, Eppendorf 5702R low-speed centrifuge, Shimadzu LC-20A high performance liquid chromatography.Human thyroid papillary carcinoma cell BCPAP was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. SS were air-dried under laboratory conditions and then ground into small pieces. About 50 g of SS was soaked in ethanol solvent (200 mL) for 7 days and filtered with cotton plugs. The ethanol is evaporated under pressure, and the extract is concentrated using a rotary evaporator. Shimadzu LC-20A high performance liquid chromatography (HPLC) with dual solvent pump high pressure gradient system, SPD-20A photodiode array detector and automatic sampler are used for one-dimensional separation. Accurately weigh the total 95% ethanol extract of SS and brucine standard respectively, and dissolve them in methanol to make the final concentration 1 mg/mL. Using 0.5 μM organic membrane filtration and preparation for HPLC analysis. The gradient of binary mobile phase composed of ethanol solvent was used for chromatographic elution. The initial gradient condition is set as 5% ethanol, the flow rate is 1.0 mL/min, and the gradient increases to 100% ethanol within 60 min. The column temperature was maintained at 37 °C during the whole process. Shimadzu C18 column , the detection wavelength is 254 nm. The volume of the total 95% ethanol extract of SS and brucine standard injection is 20 μL.2 incubator. Cell growth was observed under inverted microscope, and cells in logarithmic growth phase were taken for subsequent experiments.BCPAP cell was cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin streptomycin at 37 °C and 5% COSS respectively. Control group: the same volume of DMSO (0.01%). BCPAP cells were treated with 5 × 103 cells (100 μL suspension) was inoculated into 96 well plates. Place it in a 37 °C, 5% CO2 incubator for 24 h, and then the cell monolayer will cover the bottom of the hole. The concentration of 4 active compounds added to the cells at 2, 4, 8, 16, 32, 64, 128 µmol/L in complete medium 100 μL, each with 3 double wells. Add 90 μL serum free DMEM and 10 μL CCK8 solution per hole, continue to culture for 2 h, and then measure the absorbance of each hole with the microplate reader at 450 nm wavelength. Repeat the experiment for three times. Cells were continued to cultured for 48 and 72 h, and measured the cell activity in each group at 24, 48, 72 h respectively. Cell viability was measured by CCK8 according to the manufacturer’s instruction24. Measured the OD value of each well on the computer for the sample to be tested. The cell survival rate is expressed as a percentage, and calculate the half inhibition rate (IC50).Grouping: different concentration of drug group: 2, 4, 8, 16, 32, 64, 128 µmol/L, 4 active compounds -catechin, brucine) of 25. Western blot method was used to detect the expression levels of anti-PTC related proteins. Brucine with the final concentration of 16, 32, 64, 128 µmol/L was added to the culture dish of BCPAP cells, culture for 48 h, collect cells on ice, extract the total protein of each group of cells by RIPA lysis buffer assay kit. Measure the protein concentration by BCA method, gradually carry out sample loading, electrophoresis, membrane transfer, membrane washing and sealing. Add the corresponding primary antibody  in proportion of 1:1000 respectively, incubation at room temperature for 1 h according to instructions, overnight at 4 °C, add the secondary antibody (IgG-HRP-conjugated antibodies) after membrane washing, incubate in a shaking table for 2 h, and wash the membrane with TBST for 3 times, 10 min each time. ECL imaging system is used for luminous development. The experiment is repeated at least 3 times, and the gray value is measured and statistically analyzed.Protein blotting is a gold standard method used to identify and quantify specific proteins in complex mixtures extracted from cell or tissue lysatesP < 0.05 was considered as statistically significant difference.The data obtained were expressed in x ± s, analyzed by SPSS 23.0 statistical software, compared between groups by one-way ANOVA, and the images were drawn by GraphPad Prism 9.0 software. Image J software was used for protein gray scale calculation, and No patient involved.SS are determined  of each compound retrieved and downloaded from PubChem database was verified by molecular docking with the first 6 selected core targets, and the Discovery Studio 2019 software was used for analysis. The libdock scores was showed in Table ets Fig. A and theets Fig. B were prets Fig. A–D, stigets Fig. E–H, and ets Fig. I–L.TableOnline survival analysis showed that the expressions of IL-6 (HR = 1.6), VEGFA (HR = 1.4), JUN (HR = 1.4), TP53 (HR = 1.9) and PTGS2 (HR = 1.3) were significantly correlated with the overall survival of PTC respectively. There was no significant correlation between the expression of 1L1B HR = 0.88) and overall survival -catechin at 24, 48 and 72 h were 0.448, 0.377 and 0.351 µmol/mL respectively . Therefore, brucine was selected as the most effective compound of SS in subsequent experiments. It was revealed that brucine has a significant dose effect relationship on the inhibition of BCPAP cells but without time effect relationship, and the inhibition rate is the highest.The active compounds brucine, isobrucine, (+)-catechin and stigmasterol from ely Fig. A. The ICely Fig. B. The ICely Fig. C. The ICely Fig. D. Among SS on anti-PTC, the protein expression levels of IL6, VEGFA, JUN, TP53, 1L1B and PTGS2 were detected in this study  of brucine -catechin from 30. It was also revealed that isobrucine may make a contribution to the anti-proliferation on HepG2 cells, although they are present in SS with small quantities31. Previous research found that isobrucine showed the potential anti-cancer activities of tumor cell lines of K562, HeLa and HEP-232. Recent studies on stigmasterol rich plant extracts have shown that they have significant anticancer effects on various tumor cell lines by inhibiting cell cycle progression and inhibiting cell growth by regulating cell proliferation. Especially in skin cancer33, gastric cancer34, lung cancer35, and liver cancer36, stigmasterol plays a prominent role through different mechanisms, and its activity seems to be dose dependent. Catechin is natural phenolic compound which is a phytopharmaceutical with promising anticancer effects but poor bioavailability37. Catechin have shown effectiveness as anti-inflammatory and anti-cancer, mainly through its activity to alter the pathway by NF-ΚB, Nrf-2, and MAPKs38. Compared with chemotherapy drugs, these catechin nanoparticles have relatively low systemic toxicity and are promising drugs with low side effects for cancer treatment. The anticancer activity of catechins has been demonstrated in various in vitro and in vivo cancer models with different potential molecules40. Taken together, above 4 active compounds of SS showed relatively strong broad-spectrum anti-cancer activity.In addition, isobrucine, stigmasterol, (+)-catechin the three potential pharmacological components may also play a synergistic anti-tumor effect, which deserves further experimental study. It was reported that isobrucine exhibits most potent cytotoxicity to tumor cell lines of MCF-7SS. Through the construction of SS “drug-component-disease” target network and target gene PPI network, it was revealed that the target sites coordinated the process of proliferation, apoptosis, differentiation and metabolism to inhibit tumor metastasis through interaction. It has been demonstrated that the IL6 blockade potentiates the anti-tumor effects of γ-secretase inhibitors in Notch3-expressing can be abrogated by the IL6R blocking antibody tocilizumab in breast cancer41. It was also suggested that mucoepidermoid carcinoma patients might benefit from combination therapy with an inhibitor of IL-6R signaling and chemotherapeutic agent such as paclitaxel42. In previous study, it was revealed that IL-6 exhibits a significant role in thyroid cancer progression and targeting IL-6 signalling may be helpful in clinical management of thyroid carcinoma (TC) patients with more aggressive tumour characteristics43. It was showed that the expression of CD30L/CD30 is accompanied by the expression of IL-6/IL-6R signal. It may be of clinical significance to evaluate the expression of IL-6 protein in PTC and MTC, because of the expression level is related to tumor invasiveness44.Through network pharmacological research, 6 key targets including IL6, VEGFA, JUN, TP53, 1L1B and PTGS2 were screened as important anti-PTC targets of 46. Previous research found that VEGFA gene and its protein product is widely expressed and increased in PTC and colloid goiter45. In addition, it was revealed that the molecular status of VEGFA may play a significant role in the progression of PTC46. JUN protein may be used as a specific diagnostic biomarker/therapeutic molecular target of PTC47. It was reported that the c-Jun was associated with the presence of extra-thyroid invasion and degree of tumor infiltration, while the T allele and acetylated c-Jun also correlated with tumor stage48. P53 gene is a kind of tumor suppressor gene. It is a negative regulator in cell growth cycle, and is related to cell cycle regulation, DNA repair, cell differentiation, apoptosis and other important biological functions. One of the most powerful obstacles to cancer is the normal function of p53. As a tumor suppressor gene, TP53 gene is a ubiquitous mutant gene, which may be involved in the formation and development of thyroid cancer49. Previous studies indicated that, the prevalence of homozygous Arg TP53 genotype in adult patients with radiation related PTC is significantly reduced compared with sporadic PTC cases and the general population, which suggests that other TP53 allele combinations may lead to the risk of papillary thyroid cancer in individuals with late childhood exposure50. IL1B is a key mediator for PTC to increase the release of immune regulatory factor prostaglandin E2 and tumor necrosis factor induced gene 6 protein and the expression level of various chemokine genes51. It was reported that IL1B polymorphism is risk factor for thyroid carcinoma in a Chinese Han population52. In previous study, it was found that the mRNA levels of PTGS2 encoding prostaglandin-endoperoxide synthase 2 increased in PTC, and an increased consumption of arachidonic acid was observed, which forms the oncogenic lipid in PTC53. It has been demonstrated that PTGS2 gene is detected in a large part of human thyroid cancer, which highlights the possibility of inhibiting tumor growth through COX-2 inhibition54. In the present study, we revealed that the expressions of IL6, VEGFA, JUN, TP53, 1L1B and PTGS2 were decreased by regulated by increase of the dose of brucine, isobrucine, stigmasterol, and (+)-catechin respectively.VEGFA protein has the biological activity of inhibiting the formation of new blood vessels. When tumor cells appear, its expression level will generally rise greatly. Some studies have shown that VEGFA is related to tumor infectivity and tumor susceptibility55. Previous research revealed that the pathogenesis of cancer is closely related to cell apoptosis56. Caspase-3 protein is one of the most important apoptotic executors among the caspase family and a major effector factor in the process of cell apoptosis. Its activation marks the irreversible stage of apoptosis57. Caspase-8 protein plays an important role in the development and progression of cancer, activating various structural and regulatory proteins involved in caspase-3 division58. These proteins are crucial for cell survival and maintenance, mediating and amplifying cascade reactions during apoptosis. It was reported that in the mitochondrial apoptosis pathway, when it receives a signal that is activated, it will cause cyto-c to enter the cytoplasm, bind with apaf-1 to form an apoptotic body, and induce caspase-9 activation. Finally, caspase-9 will transmit apoptosis information to the apoptotic executing protein caspase-3, triggering an apoptosis response59. Previous studies have shown that the bad protein is expressed in various cells and can participate in the entire process of cell apoptosis through cell signal transduction pathways and interactions with members of the caspase family60. In addition, as an anti-apoptotic factor, the physiological function of bcl-2 is to suppress cell apoptosis and prolong cell life. Related research shows that bcl-2 overexpression in cells can lead to changes in the nuclear redox balance, which plays a protective role by causing the accumulation of glutathione in the nucleus and reducing the activity of caspase61. In the present study, we further investigated the protein expression levels of BAD, BCL2, CASP3, CASP8, and CASP9, respectively. The results showed that brucine may significantly reduced the protein expression levels of BCL2, CASP3, CASP8, CASP9, and significantly increased the protein expression level of BAD. Furthermore, it was reported that cleaved caspase is the activated form of caspase which usually exists in cells undergoing apoptosis62. Therefore, the cleaved CASP3, cleaved CASP8, and cleaved CASP9 were detected respectively. The results revealed that brucine may increase the protein expression levels of BAD, cleaved-CASP3, cleaved-CASP8, and cleaved-CASP9 in BCPAP cells.Apoptosis refers to the programmed death of the body under the macro regulation of genes to maintain the stability of the internal environment of cellsSS anti-tumor related targets showed that multiple signal pathways were related to anti-tumor, including IL-17 signaling pathway, endocrine resistance pathway, breast cancer, prostate cancer, etc. Through comparison, it was found that the most important pathways related to anti PTC mainly including TNF signaling pathway and MAPK signaling pathway. Previous researches showed that the representative endocrine resistance63, TNF signaling pathway64 and MAPK signaling pathway65 play an important role in the intracellular signal transduction pathway at all stages of tumor genesis and development, not only mediating cell activity through their own signal transduction process, but also jointly mediating complex biological activities in cells. The biological processes involved in the anti-PTC target of SS contain response to xenobiotic stimulus, mononuclear cell differentiation, positive regulation of transcription from RNA polymerase II promoter involved in cellular response to chemical stimulus, regulation of transcription from RNA polymerase II promoter in response to stress etc. Through the GO biological process enrichment analysis and KEGG signal pathway analysis of the anti-PTC related targets of SS, it has demonstrated that SS exerts its anti-tumor effect through multiple targets and multiple pathways.Further analysis on GO biological process enrichment and KEGG signal pathway of 67. In the present study, it has been demonstrated that the 4 active components of SS, isobrucine, stigmasterol, (+)-catechin and brucine, could bind to the 6 core protein targets in varying degrees. Experimental verification mainly concluding CCK8 test and Western blot test. CCK8 test results showed that brucine could significantly reduce the activity of BCPAP cells compared with isobrucine, stigmasterol, (+)-catechin. Western blot result showed that brucine could reduce the protein expression levels of IL-6, VEGFA, JUN, TP53, 1L1B, PTGS2, BCL2, CASP3, CASP8, CASP9 while increase the protein expression levels of BAD, cleaved-CASP3, cleaved-CASP8, and cleaved-CASP9 in BCPAP cells as the increase of dose, respectively.Molecular docking is a method of drug design based on the characteristics of receptors and the interaction between receptors and drug molecules. In recent years, molecular docking has become an important technology in the field of computer-aided drug researchSS on anti-PTC was studied by screening the active components of SS and predicting the anti-tumor target, constructing the “component-disease-target” network diagram, constructing the PPI network, and conducting GO, KEGG analysis and other network pharmacological methods on the target. It was successfully predicted the main active components of SS anti-PTC effect, anti-PTC target and its related signal pathways and biological processes. It reflects the complex mechanism of multi-component and multi-target effects of TCM on diseases. To our knowledge, this is the first application of in silico molecular docking to identify a small molecular and compounds from SS in treatment of PTC-related cells. At the same time, the manuscript also studied the expression of active ingredient brucine in SS on the target protein through experimental verification methods, proving that the active ingredient may act on PTC through related pathways, which provides a research basis for SS anti-PTC.In the present study, the molecular mechanism effect of Supplementary Information.


Chronic wounds in diabetic patients can take months or years to heal, representing a great cost for the healthcare sector and impacts on patients’ lifestyles. Therefore, new effective treatment alternatives are needed to accelerate the healing process. Exosomes are nanovesicles involved in the modulation of signaling pathways that can be produced by any cell and can exert functions similar to the cell of origin. For this reason, IMMUNEPOTENT CRP, which is a bovine spleen leukocyte extract, was analyzed to identify the proteins present and is proposed as a source of exosomes. The exosomes were isolated through ultracentrifugation and shape-size, characterized by atomic force microscopy. The protein content in IMMUNEPOTENT CRP was characterized by EV-trap coupled to liquid chromatography. The in silico analyses for biological pathways, tissue specificity, and transcription factor inducement were performed in GOrilla ontology, Panther ontology, Metascape, and Reactome. It was observed that IMMUNEPOTENT CRP contains diverse peptides. The peptide-containing exosomes had an average size of 60 nm, and exomeres of 30 nm. They had biological activity capable of modulating the wound healing process, through inflammation modulation and the activation of signaling pathways such as PIP3-AKT, as well as other pathways activated by FOXE genes related to specificity in the skin tissue. Exosomes 50–150 nm) produced by plants and animals  50 nm pro. They poNon-healing wounds greatly affect a patient’s quality of life, and represent an important economic burden. In the United States alone, they account for approximately USD 50 billion in healthcare costs each year, while scars from surgical incisions and trauma account for nearly USD 12 billion and burns account for USD 7.5 billion each year. Several therapies for wound healing are partially effective [Chronic wounds can take months or years to heal, and are characterized by high levels of inflammatory cytokines, loss of angiogenesis, and defective macrophages, etc. . PeripheIMMUNEPOTENT CRP (ICRP) is a mixture of low molecular weight substances that are less than 12 kDa, and is obtained from the dialyzable extract of bovine leukocytes from the spleen. It has been shown to be capable of modifying the immune response by regulating inflammation and inducing an antioxidant effect . It has Splenic stem cells of Hox11 lineage can differentiate when injuries exist in pancreatic islet tissues . Thus, tBased on the diversity of biological functions of ICRP, there have been research efforts to identify compounds responsible for each function. Nevertheless, there have not been conclusive results, except that ICRP has greater antitumoral activity when used as a complete spleen dialyzed extract. However, new biological applications can be elucidated more clearly when their peptides are identified and analyzed. Furthermore, previous studies using proteinase-K treatment on the whole extract increased antitumor and antioxidant activities in vitro . Additiop < 1 × 105; moreover, they had catalytic activities, such as hydrolase and transferase activity, with the same p-value. Both the proteomic and AFM analyses confirmed the presence of exosomes exerting biological functions.ICRP contains exosomes capable of surviving the freeze-drying process of the commercial product. They maintain a semi-spherical shape of approximately 60 nm . Due to p < 1 × 107 to 1 × 109) in p < 1 × 105 to 1 × 107)  and cornification (5.98 × 106) values are shown in q-value (correction of the random error) in the correction of the p-value using the Benjamini–Hochberg logarithm, with only 2% chance of having false positives for the keratinization process and 2.25% for cornification  . Based ofication .p > 0.05 diminishing false positives, as observed in These pathways coincide with the ones shown by Panther ontology (Fisher’s Exact test) , Table 2p = 3.05 × 106 for both)  in the ICRP extract, exosomes appeared to be secondary (0.0000178 for keratinization) but within same color scheme (p = 0.00000598 for ICRP and p = 0.00000305 for exosomes). For exosomes, the pathways most strongly induced were granulocyte activation (p = 0.00000222), neutrophil activation (p = 0.00000222), myeloid leukocyte activation (p = 0.00000234), and cornification, as mentioned   and cornification (0.00000183)  . Whilst r scheme . Neverthentioned . For Mus0000633) a, which and ICRP b. Nevert0000183)  processe0000183) . This dep-value score for said process  are collected and grouped into groups based on their similarities of membership (subtrees with similarity greater than 0.3 are considered a group) and corrected using the Benjamini–Hochberg algorithm. This analysis coincides with some predictions made in GOrilla and Panther, such as regarding neutrophil degranulation and cellular response to stimulate angiogenesis at different levels . It is worth highlighting that the PID-ILK pathway mentioned in According to the Panther Classification System, peptides with an abundance of exosomes could interact with components of pathways relevant to wound healing, such as angiogenesis, the EGF receptor signaling pathway, the endothelin signaling pathway, the FGF signaling pathway, and the VEGF signaling pathway a, as wel process ; those dt levels . As obsep-value, locating the closest pathways and with the highest value to the center and right side of the scheme, this being the pathways’ cellular response to stimuli, supramolecular fiber organization, the regulation of the actin cytoskeleton, the regulation of the protein-containing complex assembly, translation, and the formation of a cornified envelope (p = 1 × 1010 to 1 × 1020). Moreover, peptides have more interaction in skin tissue (p = 1 × 1015) (p = 1 × 107), with a remarque expression of FOXE1 target genes (p = 1 × 1020)   and seem × 1020) .For this reason, the PIP3-AKT signaling pathway was analyzed. In Homo sapiens predicted the intended final destiny of the product in the keratinizing and cornifying functions and the processes of cellular response to chemokines, neutrophil degranulation, the regulation of intracellular estrogen receptor, and the regulation of fat cell differentiation as secondary functions with statistical significance in processes, matching with the putative functions of the other program. Panther ontology for Bos taurus prediction also showed similar values for Homo sapiens, as observed in Exosomes can be secreted by all cells in the body, and thus we hoped to observe their presence in a spleen extract such as IMMUNEPOTENT CRP, derived from a variety of cells that arrive in that organ. The presence of exosomes in IMMUNEPOTENT CRP was demonstrated by AFM and matches with the size range for an exosome, between 50 and 200 nm ,16, showD. melanogaster cell repair processes, Rho has been reported to be required for the activation of myosin II, leading to its association with actin, which functions in the formation of actin filaments [p-value = 0.00000598, and for exosomes p-value = 0.00000305), which means with higher enrichment in exosomes than in the whole extract. When the proteins overexpressed from 6.06% to 100% in exosomes were examined, the functions outstanding in the whole extract were the same for exosomes in a prediction model of Homo sapiens , increasing enrichment for the cornification process; this could imply that the biological effects exerted by the ICRP are attributed to the exosomes present in the extract, although a potency test is necessary to prove that. In addition, when cellular components of IMMUNEPOTENT CRP were evaluated, the presence of extracellular exosomes in the sample was highlighted with significant differences (sharing importance with keratin filament cellular components), for which the biological functions of the extract could be attributed to the exosomes, as those are one of the main components.In the ilaments ,23. The ilaments . HoweverMoreover, the presence of specific types of keratins supports the hypothesis that the exosomes that possess keratin filaments present in IMMUNEPOTENT CRP can induce wound healing. In the exosomes, over-expressed keratins 1 and 2 can be found with respect to IMMUNEPOTENT CRP, and they have been reported to be overproduced in the healing process  in compaIn diabetic patients, wounds become chronic easily due to the lack of control of hyperglycemia, and these patients are reported to have defects in vessel formation, besides an inefficient closure of the wound with the formation of ulcers, and an inability to control infections . The PanAn Important concern is the site of action of a treatment; in our case, the main site of action was in skin tissue in a prediction for humans, suggesting a possible treatment employing exosomes isolated from IMMUNEPOTENT CRP in diabetic foot ulcers. We hypothesized that the exosomes activate proliferation-inflammation pathways, such as PIP3-AKT, as the pathway was significantly highlighted at different points, probably through the phosphorylation of MKRN1 and BAD, ending in the activation of genes; for example, FOXO genes correlating in Reactome and METASCAPE analysis. This pathway has been reported to induce wound healing in diabetic models , resultiIMMUNEPOTENT CRP is a registered product composed of a mixture of substances of <12 kDa from spleen extract manufactured by LONGEVEDEN SA de CV, sold in a lyophilized presentation in Mexico. For proteomic analysis, a lyophilized pool was created from different batches to assure homology and representative data of the product. Then, this pool was analyzed using Tymora Analytics Operations  to identify the proteins and digested to prepare a sample for liquid chromatography coupled to mass spectrometry coupled in tandem (LC-MS/MS), as described by Wu et al. . The comFrom the pool of ICRP sent to Tymora Analytical Operations Company , a part of this mixture was separated to isolate the exosomes through EVtrap methodology with automated magnetic bead separation. After that, they were lysed to liberate protein content. Then, proteins were extracted and digested in an SDC buffer, and later proteins were identified through LC-MS/MS analysis as specified by Wu et al. . This li2 image sizes were captured systematically for each sample at three different regions at least. The samples were analyzed with Gwyddion version 1.6 software to observe the morphological aspect of the exosome [Exosomes were isolated from a pool from different batches of lyophilized ICRP employing the kit Exoquick  that separates extracellular vesicles through ultracentrifugation. After that, the pellet was resuspended in sterile PBS 1X and analyzed with atomic force microscopy. The samples were observed using an NT-MDT Spectrum, NTEGRA Prima AFM at room temperature, with an RTESPA probe  of spring constant k = 40 N/m in intermittent contact mode. Images of height, deflection, and phase were obtained; 20 × 20, 10 × 10 and 5 × 5 µm exosome .Homo sapiens and Mus musculus). Two simulations were performed in the target list, one including all proteins present in the exosomes (1% to 100% relative abundance) and a second one with overexpressed proteins in exosomes (6.06% to 100% relative abundance) with respect to the background list; for predictions employing the target list, the background list was introduced as reference. The same procedure was performed in Panther ontology, specifying as model organisms Bos indicus, Homo sapiens, and Mus musculus. The software gave us data for molecular functions, cellular components, and biological processes.The background and target lists were composed of the gene names that codified the proteins. First, putative biological pathway functions were predicted on the online software Gene Ontology enRIchment analysis and visuaLizAtion tool (GOrilla) using a background list and target list  that interacted with components of cell signaling pathways related to wound healing in the literature and those correlated with the results obtained in previous software, Reactome version 84 software was used, entering background and target lists in the analyze gene list section (enabling the button to show interactors in the results), and with the PADOG analysis method, introducing “compare We determined that the different biological activities of spleen cells’ dialyzed extract (IMMUNEPOTENT CRP) can in part be attributed to their total peptide content and peptides contained in exosomes, and not exclusively to a particular peptide. Furthermore, the in silico analysis coordinated with activities described before, such as anti-inflammatory capacity, but new activity, such as wound healing, was found, which could lead to new preclinical research to elucidate its activity, both regarding the effect of the total ICRP extract and its exosomes. Predictions indicated a putative route in which the ICRP could exert a wound healing process through inflammation modulation and the activation of signaling pathways such as PIP3-AKT, among others.


Most lung cancer patients worldwide  have a poor survival: 25%-30% patients die < 3 months. Yet, of those surviving > 3 months, 10%-15% patients survive (very) long. Astragali radix (AR) is an effective traditional Chinese medicine widely used for non-small cell lung cancer (NSCLC). However, the pharmacological mechanisms of AR on NSCLC remain to be elucidated.Ultra Performance Liquid Chromatography system coupled with Q-Orbitrap HRMS (UPLC-Q-Orbitrap HRMS) was performed for the qualitative analysis of AR components. Then, network module analysis and molecular docking-based approach was conducted to explore underlying mechanisms of AR on NSCLC. The target genes of AR were obtained from four databases including TCMSP  database, ETCM (The Encyclopedia of TCM) database, HERB (A high-throughput experiment- and reference-guided database of TCM) database and BATMAN-TCM  database. NSCLC related genes were screened by GEO (Gene Expression Omnibus) database. The STRING database was used for protein interaction network construction (PIN) of AR-NSCLC shared target genes. The critical PIN were further constructed based on the topological properties of network nodes. Afterwards the hub genes and network modules were analyzed, and enrichment analysis were employed by the R package clusterProfiler. The Autodock Vina was utilized for molecular docking, and the Gromacs was utilized for molecular dynamics simulations Furthermore, the survival analysis was performed based on TCGA (The Cancer Genome Atlas) database.Seventy-seven AR components absorbed in blood were obtained. The critical network was constructed with 1447 nodes and 28,890 edges. Based on topological analysis, 6 hub target genes and 7 functional modules were gained. were obtained including TP53, SRC, UBC, CTNNB1, EP300, and RELA. After module analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that AR may exert therapeutic effects on NSCLC by regulating JAK-STAT signaling pathway, PI3K-AKT signaling pathway, ErbB signaling pathway, as well as NFkB signaling pathway. After the intersection calculation of the hub targets and the proteins participated in the above pathways, TP53, SRC, EP300, and RELA were obtained. These proteins had good docking affinity with astragaloside IV. Furthermore, RELA was associated with poor prognosis of NSCLC patients.This study could provide chemical component information references for further researches. The potential pharmacological mechanisms of AR on NSCLC were elucidated, promoting the clinical application of AR in treating NSCLC. RELA was selected as a promising candidate biomarker affecting the prognosis of NSCLC patients.The online version contains supplementary material available at 10.1186/s12906-023-04148-9. Lung cancer is the leading cause of cancer-related mortality. It is subdivided as Non-Small-Cell Cancers  and Small Cell Lung Cancers (SCLC), as SCLC have a very poor prognosis. The global overall 5-year survival rate of non-small cell lung cancer (NSCLC) is also less than 20% and thus poses a serious threat to public health .NSCLCs are typically divided as operable  and non-operable  at the time of diagnosis, whose life expectancy is so poor, that surgery in general is not recommended . This leUnderstanding the molecular biological mechanisms of the NSCLC cancer cells have been extensively studied and show geographic/racial variations. For example, in Asian patients, EGFR-mutation status was 51% positive, but in western countries such as Norway, it is much lower 8%). Intercellular signal pathways are also important from the therapeutic point of view. Different signaling pathways mechanisms are known, the EGFR %. Interc, Wnt 1010, and TMany patients in Asia use Traditional Chinese Medical (TCM) herbs, also because of the serious side effects of western cytotoxic drugs. Indeed, TCM herbs additional to widely used platinum-based chemotherapy, strongly reduces nausea and vomiting  and alsoAstragali radix  is a dominant herb in NSCLC anticancer treatment exhibits immunomodulatory, anti-oxidant, anti-inflammatory and anti-viral properties and shows strong antitumor effects in various tumors including NSCLC , 16. It Astragaloside IV is one of the main active components from AR, which has attracted much attention in the field of antitumor in recent years . It was Network pharmacology, proposed by Li , provideWe therefore have applied Network Pharmacology to unravel the working mechanism of Astragali Radix (Huangqi), in Non-Small Cell Lung Cancer. Moreover, Molecular Complex Detection (MCODE ) was appWe firstly employed ultra-high performance liquid chromatography-quadrupole/Orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) to identify the components absorbed in blood of AR. Secondly, we collected information about components, component-related targets, and NSCLC–related genes from extensive databases and identified crucial targets and key pathways based on network module analysis and molecular docking. Finally, the prognostic value of AR-NSCLC hub genes was analyzed using bioinformatics technology. The detailed flowchart of this study is summarized in Fig. SPF grade male Sprague–Dawley (SD) rats  were purchased from SPF (Beijing) Biotechnology Co., Ltd. . All rats were housed in the specific-pathogen-free grade animal observation room of Chengdu University of Traditional Chinese Medicine , which was set at 21 ± 1℃ and 60% relative humidity with a 12 h light/dark cycle. Rats were deprived of food for 12 h before the start of experiment, but permitted of free access to drinking water. This study was performed in accordance with the guidelines of the National Health Institutes of China and approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine .Astragali radix was obtained from affiliated hospital of Chengdu University of Traditional Chinese Medicine. Briefly, astragali radix was soaked in 8-fold mass of purified water for 30 min and then decocted for 50 min twice. After being filtered with gauze, the two filtrates were combined and concentrated to 1 g/mL using a rotary evaporator at 40 °C for intragastric administration.After 7 days of acclimation, SD rats were randomly distributed in two groups including control and AR group (2.7 ml/100 g), nine rats in each group. The control group was given equal volume distilled water.Blood was collected at 30,60 and 120 min after administration, and then the rats were anesthetized with pentobarbital sodium. Blood samples of 3 rats were collected each time, then the rats were dragged by the neck to death after the experiment. The collected blood was centrifuged for 15 min at 4000 r/min, 4 ℃. 250 µL plasma was mixed with 1000 µL acetonitrile  and performed vortex for 4 min, then centrifuged for 15 min at 13,000 r/min, 4 ℃. The supernatant was transferred to another clean Eppendorf tube, and evaporated until dry using a mild nitrogen gas stream at 25 ℃. Add 150 µL 0.01% hydrochloric acid methanol (1:1) mixed solution for redissolution, centrifuge at 13,000 r/min, 4 ℃ for 15 min, and suck the clear solution into the tube. The plasma test samples collected at three time points in the same administration group were mixed and filtered through a 0.22 µm filter for UPLC-Q-Orbitrap HRMS analysis.20 ml of AR decoction was weighed accurately, added with 100 ml of 75% methanol solution, and weighed accurately again. After refluxing for 60 min, 75% methanol solution was used to make up the lost weight and then filtered. 90 ml of filtrate was evaporated using a rotary evaporator at 40 °C. The dried extracts were redissolved in methanol to 10 mL. The redissolved methanolic solution was filtered with a 0.22 μm filter before injecting into the UPLC-Q-Orbitrap HRMS system.TMC18  eluted with a mixture of 0.1% formic acid (A) and acetonitrile (B). The proportion of acetonitrile (B) was increased from 1–20% (0–8 min), 20–30% (8–10 min), 30–35% (10–12 min), 35–40% (12–14 min), 40–55% (14–23 min), 55–70% (23–27 min), 70–80% (27–30 min), 80–99% (30–37 min), 99% (37–38 min). The flow rate was 1 mL min − 1, and the column temperature was set at 40 °C. The injection volume was 5 μL.The qualitative analysis of chemical components was performed using Ultra Performance Liquid Chromatography (UPLC) system coupled with Q-Orbitrap HRMS/MS . The sample was separated by using Thermo Scientific AccucoreMass detection was performed on a Q-Exactive Orbitrap Mass Spectrometer equipped with an Electron Spray Ionization source in positive and negative ion mode. Mass conditions were as follows: the spray voltage in positive/negative mode was 5.0 kV/3.0 kV; sheath gas (N2) flow rate of 50 L/h Pa; and the probe heating temperature is 350 ℃. In the automatic MSE mode, the CLT-GND voltage (+) and CLT-GND voltage (-) are 20 V and 15.9 V respectively. The full-scan mass spectrum was recorded in m/z 50–2,035 Da. All data were acquired and processed by Compound Discoverer 2.9 software .https://tcmspw.com/) [http://www.tcmip.cn/ETCM/index.php/Home/Index/) [http://herb.ac.cn/) and BATMAN-TCM [http://bionet.ncpsb.org.cn/batman-tcm/). A union of the search results was used to establish an AR related target genes.Four database were used to search AR’s components targets: TCMSP  database (pw.com/) , ETCM (T/Index/) , HERB [2/Index/)   obtained from the gene expression profiles including GSE1987 , GSE3353selected . The AR-http://string-db.org/) to obtained the protein–protein interactions (PPI) used for constructing the AR-NSCLC PIN. The PPI confident score was set at 0.9 to ensure the credibility of the results. Meanwhile, we performed the network analysis by means of Analyze Network plugin in Cytoscape to calculate the betweennesscentrality, closenesscentrality and degree of each node in AR-NSCLC network [Based on the AR-NSCLC shared target genes, a component-target network was constructed using Cytoscape version 3.9.0 . Then, w network . FinallyTopological properties have become very popular to gain an insight into the organization and the structure of the complex networks . TherefoCytoHubba plugin in Cytoscape was employed to calculate the degree, closeness and betweenness of nodes in critical AR-NSCLC PIN. We select the nodes as the hub genes whose score of degree, closeness and betweenness was in the top 10. Module analysis was performed by the MCODE plugin in Cytoscape to explore the significant modules in critical AR-NSCLC PIN. The advanced options set as degree cutoff = 2, K-Core = 2, and Node Score Cutoff = 0.2. Enrichment analysis, including GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis, was performed for the genes in modules to reveal the underlying mechanism through biological processes, cellular components, molecular function and key signaling pathways. ClusterProfiler package (version: 3.18.0) in R was employed to analyze enrichment analysis. The box plot was implemented by the R software package ggplot2; PCA graphs were drawn by R software package ggord; the heat map is displayed by the R software package pheatmap. All the above analysis methods and R package were implemented by R foundation for statistical computing (2020) version 4.0.3.https://pubchem.ncbi.nlm.nih.gov/). The 3D structure of protein was downloaded in RCSB PDB database (https://www.rcsb.org/). ChemBio 3D software was used to calculate and export the minimized energy stricter. Then the protein receptors were performed to remove water molecules, separate proteins, add nonpolar hydrogens, calculate Gasteiger charges of the structures in AutoDock Tools 1.5.6, and saved as PDBQT files. PyMOL 2.3.0 software was used to set the active pocket sites where molecule ligands bind and visualize the docking results. In addition, analyze all hydrogen bonding and hydrophobic interactions with ProteinPlus [The molecular docking was performed in Autodock Vina software to evaluate the accuracy of network analysis results. We downloaded the 2D structure for the molecule ligands from the PubChem database  which has generated comprehensive, multi-dimensional maps of the key genomic changes in various types of cancers. RNA-seq data (level 3) including 1017 NSCLC tissues and 108 normal tissues, and corresponding clinical information of NSCLC were obtained.To see whether hub genes were related to prognostic significance, survival analysis was performed in the R environment using TCGAbiolinks . The genP value, HR and 95% CI of each variable through ‘forestplot’ R package. Thereafter, nomogram was developed based on the results of multivariate cox proportional hazards analysis to predict the 5-year overall recurrence. The nomogram provided a graphical representation of the factors which could be used to calculate the risk of recurrence for an individual patient by the points associated with each risk factor through ‘rms’ R package. The predictive accuracy of the nomogram was assessed by calibration plot [p-values were less than 0.05.Univariate and multivariate Cox regression analyses were performed to identify prognostic factors for NSCLC. The forest was used to show the ion plot . Null hyThe total ion chromatograms of AR and AR absorbed in blood in the positive and negative ion mode is shown in Fig. An overall 688 AR components target genes were gained after removing duplication Fig. A. 294, 2The component-target network was visualized by Cytoscape with 467 nodes and 1244 edges  ~ k −γ (γ < 3) [−1.491 and y = 394.17x −1.062, respectively. So, they were a scale-free network. Small world networks have a property that mean path length is short [Biological networks have been proposed to have scale-free topology whose degree distribution follows a power law distribution  (γ < 3) . As showis short . As show P-value < 0.05 and q‐value < 0.05. The bubble plot of the top 10 KEGG pathways of each module was shown in Fig. After the intersection calculation of the nodes in the top 10 of degree, closeness and betweenness, 6 hub target genes were obtained including TP53, SRC, UBC, CTNNB1, EP300, and RELA. 7 functional modules with a score > 5 were obtained after performing the MCODE Fig. B. KEGG eP < 0.05), which was selected as the prognostic marker for NSCLC . Other clinicopathologic variables including age, pT_stage, pN_stage, and pM_stage associated with poor survival (P < 0.05). Gender, race, and smoking did notcorrelate with the prognosis of NSCLC (P > 0.05). At multivariate analysis, RELA remained independently associated with OS, with a HR of 1.43132 , along with age, pT_stage, pN_stage, and pM_stage  bound to Astragaloside IV. The RMSD curve represents positional deviations in the protein. The RELA-Astragaloside IV complex average RMSD value is 0.240771882; the EP300-Astragaloside IV complex average RMSD value is 0.189652057; the SRC-Astragaloside IV complex average RMSD value is 0.283678912; the TP53-Astragaloside IV complex average RMSD value is 0.283678912. As can be seen from Fig. In addition, the binding energy of RELA-Astragaloside IV was -37.09 kcal/mol, that of TP53-Astragaloside IV complex was -61.020 kcal/mol, EP300-Astragaloside IV complex was -33.016 kcal/mol, SRC- Astragaloside IV complex was -62.96 kcal/ mol. Molecular dynamics simulations showed that RELA-Astragaloside IV binds tightly to EP300-Astragaloside IV.The results of the GO and KEGG enrichment for modules revealed several crucial pathways  that may explain the underlying mechanisms of AR. More interestingly, the four pathways are interrelated Fig. . It is sModule 4 was enriched in JAK-STAT signaling pathway which is closely related to multiple tumorigenesis, progression, invasion, and metastasis . It has The JAK/STAT signaling pathway promotes the cytokine-mediated cell activation in a simple and efficient way . BindingModule 5 was related to the PI3K-AKT signaling pathway mainly including PIK3CA, PIK3R1, EGFR, ERBB2 , ERBB3, KDR(VEGF receptor 2), EPHA2(Ephrin type-A receptor 2), PDGFRB(platelet-derived growth factor receptor beta), IRS1(insulin receptor substrate 1), CDK2(cyclin-dependent kinase 2), CDK4(cyclin-dependent kinase 4), CDK6(cyclin-dependent kinase 6), GSK3B(glycogen synthase kinase 3 beta), SOS1(Son of sevenless 1), HRAS(HRas proto-oncogene), KRAS.In NSCLC, the PI3K-Akt pathway has been heavily implicated in both tumorigenesis and the disease progression . PIK3CA Module 6 was closely related to ErbB signaling pathway. It included EGF , HBEGF (heparin binding EGF like growth factor), NRG1 (neuregulin-1), NRG2 (neuregulin-2), ERBB4 , SRC(proto-oncogene tyrosine-protein kinase Src), RAF1.The ErbB signaling pathway performs important role in the molecular pathogenesis of lung cancer . The patModule 7 was associated with the NFKB signaling pathway which is considered the classical, or canonical pathway of NFkB . The proIn this canonical NFkB signaling pathway, the activation of IL1R1 phosphorylated IKBKB in the IKK complex consisting two catalytic subunits (IKBKA and IKBKB) and a regulatory subunit (IKBKG), and this process is mediated by the adapter protein MYD88. Then, IKK phosphorylates IkB, thus tagging it for proteasomal degradation and freeing the NFkB subunit dimers (p50/p65). NFkB dimers translocate to the nucleus where they bind to specific DNA sequences in the promoter region of a wide array of genes . Meta-anBased on survival analysis, RELA could influence the survival of NSCLC patients. And there was a positive correlation between RELA expression and tumor grade, including pT_stage, pN_stage, and pM_stage. It has prognostic value for patients and can be used to diagnose diseases or indicate the severity of diseases. The RELA played a central role in the NFκB pathway . Lin HC’Combined with molecular simulations of RSMD, RSMF, and binding free energy data, we fouce that the RELA binds better to Astragaloside IV, and thus we performed an in-depth analysis of the RELA- Astragaloside IV complex with multiple metrics. The solvent accessible surface area (SASA) is a measure of a protein’s surface area. We determined SASA between the RELA and astragaloside IV Fig. A. The fiThe free energy landscape (FEL) of the complex of RELA -Astragaloside IV was created by combining two indicators, RMSD and R(g), as shown in Fig. Pharmacological mechanisms of AR on NSCLC were explored, promoting the clinical application of AR in treating NSCLC. UPLC-Q-Orbitrap HRMS analysis of AR which provided chemical component information references for further researches. Based on network pharmacology approach, TP53, SRC, UBC, CTNNB1, EP300, and RELA were identified as hub genes. KEGG results showed that AR may produce curative effects on signaling pathway JAK-STAT, PI3K-AKT, ErbB, and NFkB. Of these hub genes, TP53, SRC, EP300, and RELA participated in the above signaling pathways and had great docking energy with the key components of AR. The survival analysis revealed that RELA was proved to have prognostic value on NSCLC patients’ survival. These findings provided references for further researches.Additional file 1: Supplementary Table 1. Pubchem compound information of drugs.


Tripterygium wilfordii Hook. F., and related studies are underway.Triptonodiol is a very promising antitumor drug candidate extracted from the Chinese herbal remedy To explore the mechanism of triptonodiol for lung cancer treatment, we used network pharmacology, molecular docking, and ultimately protein validation. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis were performed through the David database. Molecular docking was performed using PyMoL2.3.0 and AutoDock Vina software. After screening, the major targets of triptonodiol were identified for the treatment of lung cancer. Target networks were established, Protein–protein interaction (PPI) network topology was analyzed, then KEGG pathway enrichment analysis was performed. Useful proteins were screened by survival analysis, and Western blot analysis was performed.Triptonodiol may regulate cell proliferation, drug resistance, metastasis, anti-apoptosis, etc., by acting on glycogen synthase kinase 3 beta (GSK3B), protein kinase C (PKC), p21-activated kinase (PAK), and other processes. KEGG pathway enrichment analysis showed that these targets were associated with tumor, erythroblastic oncogene B (ErbB) signaling, protein phosphorylation, kinase activity, etc. Molecular docking showed that the target protein GSK has good binding activity to the main active component of triptonodiol. The protein abundance of GSK3B was significantly downregulated in non-small-cell lung cancer cells H1299 and A549 treated with triptonodiol for 24 h.The cellular-level studies combined with network pharmacology and molecular docking approaches provide new ideas for the development and therapeutic application of triptonodiol, and identify it as a potential GSK inhibitor. With the change in living habits and the increase in global emissions, lung cancer has become the most common cancer in terms of incidence and mortality worldwide. 85 percent of lung cancers in the histologic classification are non-small-cell lung cancers and share similar biological features. Due to the lack of early diagnostic techniques and the nonspecific or even asymptomatic nature of early clinical symptoms in lung cancer patients, lung cancer patients often have progressed by the time they are diagnosed . ChemothTripterygium wilfordii is a Chinese herbal medicine that is often used to treat tumors in China. Early research by our team identified one of the compounds, triptonodiol, as having excellent antitumor efficacy [Traditional Chinese medicine has a long history of application in China and plays an important role in treating tumors . After befficacy . Howeverhttps://pubchem.ncbi.nlm.nih.gov/) and the SDF format was preserved. The structural information of triptonodiol was imported into Swiss Target Prediction database (http://www.swisstargetprediction.ch/) [http://www.lilab-ecust.cn/). pharmmapper/) to obtain potential targets for triptonodiol. Lung adenocarcinoma (GSE118370) and squamous lung cancer (GSE3268) datasets were searched from the GEO database (https://www.ncbi.nlm.nih.gov/geo/), gene expression differences between tumor tissues and paracancerous tissues were analyzed, and differential genes were merged and constructed into a database of non-small-cell lung cancer disease targets.The molecule structure of triptonodiol was portrayed using PubChem  databaseion.ch/)  and Pharhttps://bioinfogp.cnb.csic.es/tools/venny/) online too, the potential targets of triptonodiol were compared with the relevant genes of lung cancer. Venn diagrams were drawn, and the intersection of the two was the potential therapeutic target of triptonodiol against lung cancer.Using the Venny 2.1 , ‘‘Organism’’ was set as ‘‘Homo sapiens’’, set the ‘‘minimum required interaction score’’ to 0.400, avoided the free target qualities, developed the PPI network [The crossing point of potential triptonodiol targets and lung cancer-related qualities was imported into STRING database   [P < 0.05 [The important genes of lung cancer and potential helpful targets of triptonodiol were imported into the DAVID , 13 dataion 8.0) , ‘‘IdentP < 0.05 , and thehttps://www.genome.jp/kegg/pathway.html) was used to search for signaling pathways and annotate the genes in the pathways. The predicted triptonodiol anti-lung cancer targets were compared with the pathway-related genes, and the intersection was taken to identify the triptonodiol anti-lung cancer enzymatic targets.The KEGG PATHWAY Database  by entering the target genes of interest and selecting tissue samples for lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC). 50% of Cutoff-High and 50% of Cutoff-Low each. The TCGA database contains expression and clinical information on 33 sorts of tumors.Survival investigation was performed on the EGPIA2 site  and A549 cells were maintained in culture using Ham's F-12K . The culture medium was supplemented with 10% fetal bovine serum  without any antibiotics. The incubator parameters were set at 37 ℃, 5% CO2, and saturated humidity.Triptonodiol was purchased from MedChemExpress  and dissolved in DMSO. The highly concentrated solution (100 mM) was stored in a − 20 °C refrigerator in a brown, lightproof vial. When required for use, dilute with culture medium to the desired concentration. The concentration of DMSO in the medium is always less than 0.1%.After COE treatment of NSCLC cells for 24 h, the cells were collected and lysed with RIPA buffer, and total cellular protein was extracted, followed by quantification using the BCA kit. Cell lysates were mixed with loading buffer and subjected to vertical gel electrophoresis on SDS-PAGE, followed by horizontal electrophoresis to transfer the proteins to PVDF (Millipore) membranes. PVDF membranes were blocked with buffer containing 10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween-20 and 5% skimmed milk, then incubated with primary antibody at 4 °C overnight , followed by incubation with a cognate secondary antibody coupled with horseradish peroxidase for 1 h at room temperature. Internal reference was controlled by GAPDH. Immunoreactive bands were detected using an enhanced chemiluminescence gel imaging system (Bio-Rad).P < 0.05, **P < 0.01, and ***P < 0.001.Statistical analysis was performed using SPSS and differences between three and more groups were analyzed using one-way ANOVA. The differences were considered to be statistically significant when *The 2D structure of triptonodiol was acquired from the Pubchem database Fig.  and enteThe potential targets of triptonodiol for lung cancer were entered into the STRING database with 16 nodes to obtain the PPI network Fig. . The nodGO and KEGG pathway enrichment analysis of the identified core targets of triptonodiol for lung cancer by DAVID software. Cellular composition and molecular functional analysis of the candidate targets were performed based on biological processes. As shown in Fig. Genetic prognostic analysis of drug targets was performed using the TCGA tumor database, which contains expression and clinical data for 33 tumors. We analyzed the prognostic impact of PKC, PAK1, and GSK3B in patients with lung adenocarcinoma and squamous lung cancer. Among these targets, GSK3B had a significant effect on lung adenocarcinoma patients, and the statistical analysis of the effect on squamous lung cancer patients was not very significant Fig. . TherefoNetwork pharmacology can significantly improve the hit rate of drug target screening, but it is still a virtual screening tool. To finally confirm the biological activity of triptonodiol against GSK3B, the protein level of GSK3B was detected by Western blot. Excitingly, the protein levels of GSK3B were significantly downregulated after 24 h of treatment of non-small-cell lung cancer H1299 and A549 with the indicated concentrations of triptonodiol Fig. . GSK3B iCurrently, tumors are one of the most serious diseases endangering human wellbeing. It is evaluated that by 2019, the number of tumor-related deaths will surpass 20 million around the world. The frequency of tumors in China is additionally on the rise year by year . ChineseTripterygium wilfordii has been used in China for at least several hundred years to treat a variety of tumor diseases, and the earliest detailed records can be found in the Qing Dynasty book “Supplement to Compendium of Materia Medica”. Tripterygium wilfordii is a renewable resource with huge reserves and has been found growing in 1219 regions worldwide [Tripterygium wilfordii, and our preliminary studies have demonstrated its antitumor effects [orldwide –24. Trip effects . In thisGSK-3 is display in all eukaryotes and could be a broadly expressed and exceedingly conserved serine/threonine protein kinase that can be inactivated by p70S6k through serine 9/21 phosphorylation –27. EMT In conclusion, we provided a preliminary exploration of the dominant targets and effective pathways of triptonodiol with the help of network pharmacology and laboratory validation of the protein molecules, confirming the antitumor pharmacological effects of triptonodiol. With the help of network pharmacology, we confirmed that ErbB signaling is the core pathway of triptonodiol's antitumor activity, which could be the basis for the development of new drugs, but more trials are still needed. Since Western blot experiments established that triptonodiol can inhibit the biological activity of GSK in vitro, we next plan to carry out animal pharmacology experiments to observe the effect and mechanism of triptonodiol for NSCLC treatment in depth and provide more references for its clinical application and development.


Our previous studies have shown that the Mubiezi-Yinyanghuo (MBZ-YYH) herb pair inhibits rheumatoid arthritis (RA) cell proliferation and glycolysis, promising results with an obscure mechanism of action.  Therefore, it is necessary to explore the main components of MBZ-YYH and unravel the potential mechanism in RA based on network pharmacology and molecular docking methods. Components and targets of MBZ-YYH were retrieved from the TCMSP. Relevant targets of RA were searched in GeneCards, therapeutic target database (TTD), and DisGeNET databases; the common targets of the MBZ-YYH compounds and RA were obtained by comparison; and a component-target interaction network was established by Cytoscape 3.9.1. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis were performed through the David database. Molecular docking was performed by PyMoL2.3.0 and AutoDock Vina1.1.2 software.  7 active ingredients and 58 putatively identified target genes were screened from MBZ, and 16 effective components of YYH and 230 potential targets were identified. There were 29 mutual targets between the two herbs and RA. Through the PPI network, 9 hub targets which contain JUN, CASP3, PPARG, PTGS2, GSK3B, CASP8, HMOX1, ICAM1, and HK2 were screened out. GO term enrichment analysis indicated that positive regulation of the apoptotic process, response to drugs, and response to hypoxia were significantly enriched. Based on KEGG analysis, it was mainly associated with the IL-17 signaling pathway, the TNF signaling pathway, and the p53 signaling pathway. The docking analysis revealed that the effective components showed strong binding activity with the receptors.  The effects of the MBZ-YYH herb pair on RA were coordinated by the interaction of diverse components, which may be through the IL-17 signaling pathway and the TNF signaling pathway, which target GSK3B, HK2, caspase 3, and caspase 8, inhibiting the proliferation and glycolysis of rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) and tending towards an increasing efficacy and decreasing toxicity effect on RA. Rheumatoid arthritis (RA) is a chronic, inflammatory, and autoimmune disease characterized by inflammatory changes of synovial tissue, cartilage, and joint skeleton . The comWith developments in the field of traditional Chinese medicine (TCM), the treatment of RA has entered a stage of diversified comprehensive treatment. As an assistant and substitute medicine, TCM has gained increasing attention because of significant efficacy and lesser side effects. Our previous studies found that an extract of MBZ could suppress the angiogenesis of human umbilical vein endothelial cells (HUVEC) in the RA synovial cavity by inhibiting glycolysis, but the specific mechanism is still unclear . MBZ hasHuman rheumatoid arthritis synovial fibroblasts (HFLS-RA) were purchased from Beina Chuanglian Biotechnology Co., Ltd.; fetal bovine serum (Sciencell); RPMI 1640 medium (Gibco); cell counting kit-8 (Beijing Solarbio); glucose content detection kit, ATP content detection kit, lactic acid content detection kit, and hexokinase activity detection kit (Beijing Solarbio).4/mL in Dulbecco's modification of Eagle's medium with 10% fetal bovine serum, and 100 μL per well was added to a 96-well plate and adherent cultured overnight. The mixture of MBZ and YYH  and cultured for 24, 48, and 72 hours. CCK-8 (10 μL) was added, and the absorbance was detected at 490 nm by a microplate reader.HFLS-RA in the logarithmic growth phase was collected by centrifugation, counted, and diluted to 1 × 10's study ) was dilμmol/104 cells) = 0.002 × (measuring tube−blank tube)/(standard tube−blank tube); HK (U/104 cells) = 2.226 × ΔA; and LA (μmol/106 cells) = 0.2375 × X.Cell glucose content was measured by spectrophotometry according to the manufacturer's instructions. G (http://tcMBZpw.com/tcMBZp.php), the processes that determine the pharmacokinetic behavior of a drug compound are its absorption, distribution in the body, metabolism, and elimination from the body (ADME). The specific screening criteria were oral bioavailability (OB) ≥30% and drug‐like properties (DL) ≥0.18. “Rheumatoid arthritis” as keywords retrieval in the GeneCards database (https://www.genecards.org/), Therapeutic Target Database (TTD) (http://db.idrbla.net/ttd/), and DisGeNET(http://www.disgenet.org/) databases to obtain RA-related disease targets. The intersection between the compounds and RA disease targets was obtained by the Venn diagram.By searching for a traditional Chinese medicine (TCM) system pharmacology (TCMSP) platform (https://string-db.org/cgi/input.pl). Cytoscape 3.9.1 (https://cytoscape.org/) was used for visualization, and the hub targets were screened according to the degree value.The intersection of targets obtained by Venn diagram was regarded as the potential targets of compounds for the treatment of RA. The intersection of the other targets of MBZ and YYH was regarded as the interactional targets, and the protein-protein interaction (PPI) network of these potential targets was constructed by STRING (https://david.ncifcrf.gov/), the P value less than 0.05 was set as a condition of screening for enrichment of gene ontology (GO) function analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the key target protein. Three modules, namely, biological process, cellular component, and molecular function, were selected for mapping. Its first 10 entries were visualized as bubble plots using the ImageGP (https://www.bic.ac.cn/ImageGP/).By the DAVID database (http://www.rcsb.org/pdb) and saved in PDB format. Three-dimensional (3D) conformation of candidate compounds (https://pubchem.ncbi.nlm.nih.gov/) was downloaded from the PubChem database and saved in SDF format and then converted into a PDB format via Open Babel. Ligands and receptors were prepared by AutoDock Vina and PyMOL (v.2.3.0). The original ligand and water molecules were removed from the crystal structure of the receptor, nonpolar hydrogen was added, and the Gasteiger partial charge was calculated. Applying energy minimization and assigning atomic charges to process ligands, the active site of molecular docking is determined by the ligand coordinates in the target protein complex. The ligand is set to be flexible while the receptor is rigid. All prepared receptors and ligands were saved in pdbqt format. The affinity of the binding strength between the ligand and the target protein was assessed with AutoDock, and the conformation with the best affinity was selected as the final docking conformation and visualized in Pymol 2.3.0. The stability of systems was evaluated using the root-mean-square deviation (RMSD) of the aligned protein-ligand coordinate set calculated against the initial frame.Referring to studies in –18, the μg/ml of the MBZ-YYH herb pair at 24 h (P < 0.001)  , so we t∗∗P < 0.01) and lactic acid (∗∗P < 0.01) and the activity of hexokinase (∗∗∗P ≤ 0.001) in RA cells were recovered by the combination of MBZ and YYH, prompting that the MBZ-YYH herb pair could inhibit the glycolysis of RA cells , while the hydrogen donor (Hdon), OB, and DL were not different. Indicating that the chemical properties of the two species were obviously different, however, to some extent, there is a certain intersection between them. Therefore, it is speculated that some components of this compatibility may be mutually beneficial, while others are mutual detoxication , octanol-water partition coefficient (AlogP), and hydrogen acceptor (Hacc) were prominently different between the two herbs  was retained for target analysis to ensure accuracy. In addition, the OB values of NON , karounidiol , and MOL012372  were similar to the mean value of the OB components of MBZ, so they were selected as candidate ingredients for further analysis .There were 55 and 212 targets corresponding to the active ingredients of MBZ and YYH, respectively. To further understand the role of targets, we obtained the target intersection that was regarded as a potential therapeutic target through the Venn diagram and constructed a PPI network. The resulting PPI network was introduced into Cytoscape 3.9.1, and hub targets were selected according to the degree values which produced form network analyzer of Cytoscape . AggregaFor the purpose of comprehending the molecular functions of potential targets, GO and KEGG enrichment analyses were performed. The results showed that GO was mainly involved in protein binding, positive regulation of the apoptotic process, response to drugs, and response to hypoxia. KEGG mainly refers to EGFR tyrosine kinase inhibitor resistance, the IL-17 signaling pathway, the TNF signaling pathway, and the p53 signaling pathway Figures . It is hIn addition, we also conducted GO and KEGG analyses on 18 common targets of MBZ-YYH, and the results showed that it mainly involves G-protein-coupled receptor activity, extracellular ligand-gated ion channel activity, regulation of membrane and neuroactive ligand-receptor interaction, calcium signaling pathway, and regulation of actin cytoskeleton Figures . It is a18 active ingredients  and 9 interest proteins with the highest degree values  for molecular docking. The results showed that the binding energy of all proteins to the active components is less than −5 kcal/mol, indicating that ligands and receptors were closely bound. The strongest proteins that bind to the active ingredients are as follows: PPARG and 8-(3-methylbut-2-enyl)-2-phenyl-chromone , PTGS2-yinyanghuo C , HK2-Yinyanghuo C , -4,4,6a,6b,8a,11,14b-heptamethyl-11-methylol-1,2,3,4a,7,8,9,10,12,12a,13,14-dodecahydropicen-3-ol , JUN-Yinyanghuo A , HMOX1- 8-(3-methylbut-2-enyl)-2-phenyl-chromone , GSK3B-Yinyanghuo C , ICAM1-bessisterol , and CASP8-oleanolic acid  .Except ICAM1-bessisterol, which only has hydrophobic interaction, the other eight pairs all interact through hydrogen bond formation and hydrophobic force. Among them, PTGS2-Yinyanghuo C  is well paired, and Yinyanghuo C is most likely to combine with HK2 and GSK3B at the same time. It is worth noting that the effect of MBZ on HK2 is mainly achieved by MOL012372, MOL012375, MOL012377, MOL000263, and MOL000358 ; YYH mainly acts on HK2 through MOL003542, MOL004382, and MOL004384  .In this study, it was found that the MBZ-YYH herb pair can inhibit proliferation and activity of hexose kinase of HFLS-RA. Further, it can restore the glucose level and reduce the content of lactic acid, suggesting that the MBZ-YYH herb pair may inhibit the glycolysis of RA cells. This finding is consistent with our previous study that MBZ treated RA by glycolysis inhibition . NeverthChemistry and pharmacology analysis shows that there are obvious differences in the chemical properties between MBZ and YYH, but they overlap to some extent. It is hinted that there may be some components that mutual promotion and mutual-assistance, mutual restrain and mutual-detoxication. Herbs interaction is reflected in the fact that each component can not only act independently but also generate new modes and mechanism of action between the components after their compatibility . Among tNetwork pharmacology analysis suggested that the common hub targets of the main components of herbs and RA were JUN, CASP3, PPARG, PTGS2, GSK3B, CASP8, HMOX1, ICAM1, and HK2. GO and KEGG analyses showed that the main targets involved response to drugs, response to hypoxia, IL-17 signaling pathway, and TNF signaling pathway, suggesting that the treatment of RA by the MBZ-YYH herb pair may regulate the response of cells to drugs and the hypoxia environment through the IL-17 signaling pathway and TNF signaling pathway.During the development of RA, hypoxia alters cell biological functions by inducing mitochondrial dysfunction and promoting the switching of glycolytic pathways, leading to abnormal cell invasion and angiogenesis . Our preMolecular docking suggested that Yinyanghuo C was stably bound to PTGS2, while it combined to both HK2 and GSK3B. In addition, MOL004384 of YYH binds GSK3B with the most affinity , MOL000358 , MOL012377 , MOL012372  of MBZ, suggesting that, for GSK3B targets, these four active ingredients are likely to bind to it and regulate RA progression. It is noteworthy that the effect of MBZ on HK2 is mainly through MOL012372, MOL012375, MOL012377, MOL000263, and MOL000358 , while YYH mainly acts on HK2 through MOL003542, MOL004382, and MOL004384 . In addition, MOL012372  and MOL000263  of MBZ had the most affinity for binding to CASP3, followed by MOL004382  of YYH. The sequence of binding to CASP8 was MOL000263  in MBZ, followed by MOL004382  in YYH and MOL012377  in MBZ.The glycogen synthase kinase 3b (GSK3b), a negative regulator of glucose homeostasis, is involved in energy metabolism, inflammation, and apoptosis pathways in a cell-type and environment-dependent manner. Active GSK3b is associated with mitochondrial quiescence in Drosophila oocytes. However, B cells deficient in GSK3B showed higher metabolic activity and stronger proliferation . The curIn summary, the MBZ active ingredients were predicted to mainly cause apoptosis and act as a major actor, while the YYH active ingredients acted as a regulator of glucose metabolism and hexokinase activity, playing an adjuvant role in RA. This provides a theoretical basis for the application of MBZ combined with YYH in the clinical treatment of RA and excavates new therapeutic drugs for treatment of RA. However, it is undeniable that there are still some shortcomings in this study. Network pharmacology relies too much on the existing database, and the composition and target prediction of TCM such as MBZ may be lagging behind and the data are not comprehensive. In addition, for the drug interactions, this study only made some predictions and inferences about the indirect effects between them. For the other targets of different components, the interaction mechanism was not predicted due to its extensive involvement. Our next work will provide more empirical findings and evidence.In summary, MBZ and YYH act on RA through the interaction and coordination of different components. It may regulate the response of cells to drugs and the hypoxia environment by targeting GSK3b, HK2, caspase 3, and caspase 8 through the IL-17 signaling pathway and the TNF signaling pathway and inhibit the proliferation and glycolysis of RA FLS cells. Hence, it has a synergistic and attenuated therapeutic effect on RA.


Pleckstrin Homology) and FYVE  domain-containing proteins. The Phafin protein family is classified into two groups based on their sequence homology and functional similarity: Phafin1 and Phafin2. This protein family is unique because both the PH and FYVE domains bind to phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide primarily found in endosomal and lysosomal membranes. Phafin proteins act as PtdIns(3)P effectors in apoptosis, endocytic cargo trafficking, and autophagy. Additionally, Phafin2 is recruited to macropinocytic compartments through coincidence detection of PtdIns(3)P and PtdIns(4)P. Membrane-associated Phafins serve as adaptor proteins that recruit other binding partners. In addition to the phosphoinositide-binding domains, Phafin proteins present a poly aspartic acid motif that regulates membrane binding specificity. In this review, we summarize the involvement of Phafins in several cellular pathways and their potential physiological functions while highlighting the similarities and differences between Phafin1 and Phafin2. Besides, we discuss research perspectives for Phafins.Phafins are PH ( Living cells utilize endocytosis to take up extracellular nutrients and membrane-bound macromolecules from the plasma membrane and transport them to the cytoplasm in endocytic vesicles, where the cargo is internalized and invaginated into vesicular and tubular carriers ,2,3. Whe2, PtdInsP2, PtdInsP2, and PtdInsP3 are the seven PIPs found in animal cells. PIPs serve as identity markers of membranous compartments. For example, the plasma membrane is highly enriched in PtdInsP2, whereas PtdIns(4)P and PtdIns(3)P predominate in the Golgi apparatus and the early endosomes, respectively . . 128]. Amplex 2) . In resptivation . Activattivation .Phafin1 is also involved in autophagy . Co-overMacropinocytosis is an actin-dependent endocytosis mechanism that cells exploit to ingest extracellular fluids and soluble macromolecules. Cargo uptake can be divided into two morphologically different steps: membrane ruffling and closure of membrane ruffles. Membrane ruffling requires rigorous remodeling of the plasma membrane followed by the internalization of extracellular fluids and solutes into cup-shaped vacuoles, which then mature into large vesicles, called macropinosomes ,133. MacA recent study demonstrated that Phafin2 promotes macropinocytosis by coordinating actin organization at macropinosomes that are forming . Phafin2PtdIns(3)P binding is critical to the recruitment of Phafin2 to macropinosomes. The PtdIns(3)P-binding defective mutant Phafin2 R176A (within the FYVE domain) completely abolishes membrane association, highlighting the critical role of the FYVE domain in this process. Phafin2 relies on coincidence sensing of PtdIns(3)P by the FYVE domain and PtdIns(4)P by the PH domain during the first phase of localization to nascent macropinosomes . The polThe motor-binding protein JIP4 is recruited to the tubulating macropinosomes by Phafin2 in a PtdIns(3)P-dependent manner B. JIP4, Phafin2 is a pattern recognition receptor in the innate immune system of zebrafish. Recombinant Phafin2 proteins bind to bacterial signature molecules in vitro, such as peptidoglycans, lipopolysaccharides, and lipoteichoic acid . More imPhafin2 gene shows a high expression level in immune cells such as CD19+ B-cells and BDCA+ dendritic cells (http://biogps.org/#goto=genereport&id=79666 (accessed on 16 November 2022)), which suggests that Phafin2 may play a key role in the immune response [The response ,140. Macresponse .Phafin2 gene was identified as an estrogen-responsive gene in breast cancer (BC) cells through DNA microarray analysis of gene expression profiles. The expression profile of the Phafin2 gene in estrogen-responsive BC cells is significantly different from that in non-responsive cells. Phafin2 is one of a set of 61 genes identified through in silico metanalysis of the transcriptomes of estrogen receptor-positive and estrogen receptor-negative human BC cell lines whose expression profiles discriminate these two cellular phenotypes in BC cell lines and tumor biopsies [Phafin2 gene is differentially expressed in human hepatocellular carcinomas (HCC). The Phafin2 mRNA level is significantly higher in the transcriptome of HCC tumors than in normal livers, indicating that Phafin2 gene expression is upregulated in liver cancer [The biopsies . In addir cancer .Phafin2 gene, amplification is the most frequent type of gene alteration. The amplification frequency of Phafin2 in BC, prostate cancer, and bladder/urinary tract cancer is more than 15%. It has been proposed that overexpression of Phafin2 gives cancer cells a growth advantage, enhancing their nutrient scavenging, possibly through its involvement in macropinocytosis. Indeed, KRAS-transformed pancreatic cancer cells are not able to scavenge extracellular proteins under amino acid-limiting conditions and exhibit reduced proliferation after deletion of Phafin2 [Phafin1 gene is also amplified in various cancers [Both Phafin1- and Phafin2-expression levels were found to be upregulated in multiple cancers by screening the publicly available cBioportal cancer genome database . In the  Phafin2 . Even th cancers , it remaPhafins are PH domain and FYVE domain-containing proteins that act as adaptor proteins in apoptotic, endocytic, and autophagic pathways . Their mExtensive membrane remodeling, such as membrane trafficking and membrane fusion, is required for endocytic and autophagic pathways. Phafin2 strongly colocalizes with lysosomes and autophagosomes during autophagy . Phafin2S. cerevisiae, Pib2, stimulates TORC1 signaling and regulates lysosomal membrane permeabilization in response to ER membrane stress [Autoinhibition plays an important role in regulating protein function. Autoinhibitory mechanisms are found in many catalytic proteins  and non-catalytic proteins  ,143. We e stress . Sequence stress . Therefo


Background: The prominent mitochondrial metabolic changes of the atrium reportedly have significant impact on electrical signals and structural remodeling which play important roles in the occurrence and development of atrial fibrillation (AF). However, the mechanism is not completely known.Objective: This study was aimed to explore the mitochondrial metabolism reprogrammed in AF patients by integrating metabolomics as well as proteomics of human atrium tissues.Methods and Results: Left atrial tissue samples were harvested from 10 non-valvular AF patients and 10 matched samples from healthy donors for transplantation. In metabolomics analysis, 113 metabolites were upregulated and 10 metabolites were downregulated in AF, where multiple pathways related to mitochondrial energy metabolism were enriched. Correlation analysis between the differentially expressed proteins and metabolites identified several hub proteins related to mitochondrial function including Glycerol-3-phosphate dehydrogenase 2 (GPD2), Synemin (SYNM), Plectin (PLEC), with MCC score of 27, 17, 16, respectively, which have the most interactions with the dysregulated metabolites and ranked at the top in network string interactions scored by MCC method. All 330 differentially expressed proteins including 225 upregulated and 105 downregulated molecules were revealed and analyzed, which identified the downregulation of GPD2 (p = 0.02 and FC = 0.77), PLEC (p < 0.001 and FC = 0.71) and SYNM (p = 0.04 and FC = 0.76) in AF patients. Gene Set Variation Analysis (GSEA) showed mitochondrial metabolism-associated pathways including oxidative phosphorylation (NES: −1.73) and ATP biosynthetic process (NES: −2.29), were dramatically diversified in human AF. In GSVA, the expression levels of GPD2, PLEC, and SYNM were demonstrated to be associated with multiple metabolic pathways related to mitochondrial function  and cardiac structural and electrical remodeling , which were proven vital in the development and maintenance of AF.Conclusion: In all, this study provides new insights into understanding the mechanisms of AF progression, especially the reprogramming mitochondrial metabolism, and identifies several genes related to mitochondrial function as novel targets for AF, which may be involved in the occurrence and development of AF. Atrial fibrillation (AF) is the most common arrhythmia managed in clinical practice with 22%–26% estimated lifetime risk, and is associated with substantial morbidity and increased mortality due to the combination of altered hemodynamics, atrioventricular dyssynchrony, progressive atrial and ventricular mechanical dysfunction, and thromboembolic complications . The risetc. .etc., or the recipient has been already evaluated not suitable for receiving transplantation anymore due to worsening condition with multiple organs failure and unable to tolerate the surgery, or the recipient had died before the donor arrived. The specimens were immediately snap-frozen and stored in liquid nitrogen  were harvested from AF patients undergoing surgical ablation of AF without cardiopulmonary bypass. Subjects (10 patients) diagnosed with non-valvular  AF suffered from persistent AF for more than 1 year. For comparison, LAAs from 10 matched SR patients were collected. The controls in SR were matched by age and gender. Normal left atrial appendages were collected from healthy donors. The specimens obtained from the homograft bank had been rejected for clinic use and therefore allocated for research. As required in the research ethics, the clinical criteria for the healthy donors rejected for transplantation including failure in pairing between the donor and recipient of blood matching, nitrogen . RelatedThis study was approved by the Ethics Committee Board of Shanghai East Hospital , and performed in line with the Declaration of Helsinki.Raw proteomics data were processed using MaxQuant_2.1.0.0, Perseus 2.0.7.0 and R package MSstats 4.4.1. Raw metabolomics data were analyzed using xcms 3.18.0. The overall proteomic and metabolic changes in AF were determined by OPLS-DA on R package mixOmics . Additiohttp://www.smpdb.ca/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://home.jp/kegg/) for pathway enrichment analysis using R package MetaboAnalystR 3.2.0. Multi-omics analysis of pathways sharing both significant proteins and metabolites using MetaboAnalyst using up- or downregulated proteins and metabolites. Gene Set Enrichment Analysis (GSEA) was also used to analyze the correlations of different gene sets with GSEA 4.1.0. Gene Set Variation Analysis (GSVA) was used to study correlations using R package GSVA 1.44.5.Dysregulated metabolites and proteins were selected and mapped into the Small Molecule Pathway Database (SMPDB)  for at least 3 or 5 independent assays unless otherwise noted. All data passed the normality and equal variance before analysis. Student’s t-test was used for 2-sample comparison. One-way analysis variance (ANOVA) with Tukey etc., which have vital functions in maintaining the homeostasis of the myocardium , PLEC (p < 0.001 and FC = 0.71) and SYNM (p = 0.04 and FC = 0.76) in AF patients in AF patients : patients .Pathway enrichment analysis was then performed. For biological processes (BP) by GO analysis, DEPs were enriched in heart development, respiratory electron transport chain and actomyosin structure organization . For celp < 0.01), ATP biosynthetic process (p < 0.01) and NADH dehydrogenase complex (p < 0.01)  , which w < 0.01) .r = 0.33), cardiac muscle contraction (r = 0.2)], and various pathways relevant to myocardial electrical remodeling, including calcium signaling (r = 0.27) and ion homeostasis (r = 0.7), and metabolic pathways such as lipid metabolism (r = 0.28) and AMP activated protein kinase signaling (r = 0.46). The expression levels of SYNM showed association with pathways related to cardiac remodeling and energy metabolism , and PLEC demonstrated association with pathways related to mitochondria function .Gene Set Variation Analysis (GSVA) was then carried out to analyze the correlation of enriched gene sets and the expression of GPD2, PLEC, and SYMN . The GPDAF is a source of significant morbidity and mortality associated with heart failure, stroke, and other complications . Atrial AF-induced irregular heartbeat alters atrial hemodynamics, oxygen delivery, and energy supply, which all influence the metabolic state . Recent Metabolomics, an emerging type of omics, reflects the downstream products of gene expression and enables a more direct and accurate reflection of the pathophysiological state of an organism. Metabolomics is widely used in new drug development, disease diagnosis, and personalized therapy . ProteomRecent studies show mitochondrial metabolism, the most predominant functional site of energy metabolism in cardiomyocytes, plays an important role in the development of AF . It alsoetc. -shuttle, playing an important role in cell energy metabolism  and cardiac structural and electrical remodeling , which were proven vital for the development and maintenance of AF . MoreoveIn addition, the limitations of this study include that analysis performed with different tools or metrics has not been compared and discussed in this study and function validation based on experimental approach should be carried out in the further study.This study provides new insights into the mechanisms of AF progression, especially the reprogramming mitochondrial metabolism, and identifies multiple genes related to mitochondrial function as novel targets for non-valvular AF, which may participate in and maintain AF.