Bailing tablet, a patent Chinese medicine, is widely used to treat vitiligo in China. However, the underlying mechanism of this combined drug in treating vitiligo still remains unclear.This study aimed to investigate the pharmacological mechanism of bailing tablet in the prevention and treatment of vitiligo using network pharmacology and molecular docking.Genetic data of vitiligo and normal people were obtained by gene expression omnibus (GEO) DataSets database and GEO difference analysis was conducted to obtain differential genes. The main active compounds and corresponding target genes of bailing tablet were collected from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. Combined with the results of GEO difference analysis, the main compounds and corresponding target genes of bailing tablet in the treatment of vitiligo were screened. The network diagram of “traditional Chinese medicine compound target” was constructed by Cytoscape software. According to the differential genes, the core targets with potential therapeutic effect were searched, the protein–protein interaction network was constructed, and the key proteins were explored by topological analysis (CytoNCA). Meanwhile, the core targets were analyzed by biological process (gene ontology) and signal pathway (Kyoto encyclopedia of genes and genomes) function enrichment. Molecular docking technology was adopted to verify the combination of main components and core targets.A total of 170 active compounds and 1777 prediction targets were screened from 11 traditional Chinese medicines of bailing tablet, of which 65 active components and 68 related prediction targets were closely related to vitiligo. A total of 320 key proteins were obtained by analyzing the topological characteristics of the protein–protein interaction network, mainly including neurotrophic receptor tyrosine kinase 1, tumor protein P53, cullin 3, estrogen receptor 1, etc. The main biological processes involve oxidative stress response, cell response to reactive oxygen species, and reactive oxygen species metabolism. Bailing tablet treats vitiligo mainly by regulating immune inflammation, apoptosis, and autophagy, which involves phosphatidylinositol-4,5-bisphosphate 3-kinase Akt signal pathway, mitogen-activated protein kinase signal pathway, Janus kinase signal transducer and activator of transcription signal pathway, melanin production, and helper T cell (Th)1, Th2, and Th17 differentiation pathway, etc. Molecular docking results showed that the main components could bind to the target protein well.Based on network pharmacology and molecular docking, the mechanism of bailing tablet in the treatment of vitiligo through multicomponent, multitarget, and multichannel was deeply explored. Melanocyte death is one of the characteristics of vitiligo. Also, the pathogenesis and etiology of vitiligo are diverse, which has not been confirmed by medical research so far. Most people hold the view that the pathogenesis of vitiligo includes oxidative stress theory, autoimmune hypothesis, neural theory, inherent theory, melanocyte hypothesis of bleeding, etc. The commonly used first-line treatment methods clinically mainly include glucocorticoid, synthetic α-melanocyte-stimulating hormone, calcineurin inhibitor, vitamin D3, phototherapy, surgery, traditional Chinese medicine (TCM), etc. The short-term efficacy of Western medicine in the treatment of this disease is acceptable, while there are certain side effects in the long-term treatment, such as local skin irritation, atrophy, telangiectasia, hormone-dependent dermatitis, and so on. TCM is commonly used for its small side effects, high compliance, and stable curative effects, and its mechanism has been confirmed by modern medicine gradually.Vitiligo is a disease of acquired pigment disorder of skin and/or mucous membranes, with epidermal melanocytes missing, accompanied with skin and leukoplakia. In children and adults, the incidence rate is 0.5% to 2%.Astragalus membranaceus, Angelica dahurica, Panax notoginseng, Angelica sinensis, peach kernel, peony bark, safflower, purslane, red peony, Fang feng, and Atractylodes rhizome. Angelica sinensis, P notoginseng, and A membranaceus could replenish Qi and blood; peony bark, red peony, peach kernel, and safflower could “activate blood circulation and remove blood stasis”; Fang feng, Atractylodes macrocephala, and A dahurica could “dispel wind and activate blood circulation”; purslane has the function of “clearing heat and cooling blood.” The combination of the above drugs has the effect of “activating blood circulation and tonifying blood,” and “removing blood stasis and dispelling wind.” Bailing tablet is recommended for the treatment of vitiligo in the consensus of experts in TCM diagnosis and treatment, and its clinical efficacy has been confirmed by many studies. A meta-analysis of 17 clinical studies, including 2832 patients, reported that conventional treatment combined with bailing tablet significantly improved the total effective rate compared with conventional treatment alone, while there are no obvious adverse reactions, confirming the efficacy and safety of bailing tablet. According to modern medicine, bailing tablet can increase photosensitivity, improve the activity of tyrosinase, and improve immunity. It is reported that bailing tablet can improve local skin microcirculation and promote the production of melanocytes to reduce leukoplakia from the periphery to the center. However, the mechanism of bailing tablet in the treatment of vitiligo is not clear. Network pharmacology can systemically reveal the internal mechanism of pharmacology from multiple levels and angles through systems biology, bioinformatics, network science, and other disciplines to have a better guiding direction for clinical diagnosis and treatment.Bailing tablet belongs to a kind of patent Chinese medicine, with the functions of “activating blood circulation and removing blood stasis” and “nourishing blood and dispelling wind.” The main components of the medicine are as follows: http://www.ncbi.nlm.nih.gov) was used to retrieve the word “vitiligo.” The data, with a sample size of >20, were selected to download and the gene data of vitiligo and normal people were obtained. According to the gene expression of samples, differential gene analysis was carried out to screen the upregulated and downregulated genes in patients with vitiligo and healthy people. R software was used for analysis, and the differential genes were obtained based on the absolute value of logFC >1 and the corrected P value <.05. The top 20 upregulated and down regulated differential genes were selected to make volcano map and heat map.Gene expression omnibus DataSets in the National Center for Biotechnology Information database (http://tcmspw.com/tcmsp.php/), a database and analysis platform of Traditional Chinese Medicine System Pharmacology (TCMSP), is a platform for running the relationship among drugs, targets, and diseases. TCMSP was used to search and collect the chemical components of 11 herbs . At the same time, 2 pharmacozoological parameters of oral bioavailability ≥30% and drug (drug-likeliss) ≥0.18 were taken as the criteria. The active components with the main biological effects in bailing tablet were screened.Tcmsp (http://www.cytoscape.org/) software to construct the network diagram of “bailing tablet–active components–vitiligo disease targets.”TCMSP was used to search the DrugBank database to screen the target genes corresponding to TCM compounds. The intersection of drug target genes and disease difference genes was used to predict the core target of TCM compounds in the treatment of vitiligo. TCM, active components of TCM, and core targets were input into Cytoscape  is a database for searching known proteins and predicting PPI that includes direct physical interaction and indirect functional correlation. At the same time, we used Cytoscape to construct PPI network. Gene information was imported into Cytoscape, and HPRD, BIND, DTP, MINT, INTACT, and BIOGRID databases were retrieved through BisoGenet to obtain protein information and select internal nodes and adjacent nodes to make PPI network. CytoNCA in Cytoscape software was used for topology analysis, and the degree centrality (DC) and betweenness centrality (BC) were taken as screening criteria. The minimum value of DC was 61, the minimum value of BC was 600, and the related proteins were obtained.P value cutoff of .05 and Q value cutoff of 0.05 were taken as the standard. The R software was used for GO enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, and the first 20 enrichment results were selected to draw a bubble diagram. The top 20 KEGG results were taken as the included pathways, the target genes corresponding to each pathway were analyzed, a network diagram was made, the pathways and occurrence times of genes were explained, and the main pathways that play an important role were predicted.Gene ontology (GO) analysis is usually divided into 3 categories: biological process (BP), cell component, and molecular function (MF). Enrichment analysis database (David) was used for the 3 types of analysis.  Protein structure was collected through protein data bank database (https://www.rcsb.org/), and the 3-dimensional structure of the main components was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The above 2 data were imported into Schrodinger software for pretreatment of target proteins and compounds, then protein ligands were selected to generate lattice files, and finally, the Ligand Docking program was operated to obtain the docking score and the result diagram of molecular docking.Molecular docking technology can simulate and predict the binding mode and affinity between target and drug molecules by computer.A total of 30 samples were included, with 15 diseased skin and 15 nondiseased skin. By comparing the related genes of vitiligo in the control group and the experimental group, 7357 differential genes were found, of which 3390 were upregulated and 3967 were downregulated. It is predicted that the pathogenesis of vitiligo is closely related to the expression of these genes Fig. . Among tThrough TCMSP database, 170 eligible active components and 1777 predicted targets were screened and collected from bailing tablet. After the intersection of the corresponding target of bailing tablet and the disease difference gene, a total of 68 related prediction targets were obtained, including PTGS2, AR, RXR, CXCL10, etc. It was predicted that these may be the main targets of bailing tablet.o-methyl isomucronulatol  indicates the binding force between the compound and the target protein. The smaller the binding energy is, the better the combination of the 2 is  has the best binding ability. It is predicted that ESR1 protein may play an important regulatory role.In order to verify the accuracy of results in our study, 6 core proteins  screened by PPI network were docked by Schrodinger software, which showed good docking ability Figs. –14. AccoAs a common acquired, localized, or generalized skin depigmentation disease, vitiligo occurs at any age and any part, without conscious symptoms. TCM doctors believe that the main etiology and pathogenesis of vitiligo is “blood deficiency and wind dryness.” The treatment is mainly to tonify the liver and kidney, nourish blood, activate blood, and dispel wind. In this article, the mechanism of bailing tablet in the treatment of vitiligo was explored from a multitude of angles and levels by using “traditional Chinese medicine compound target” network, PPI network, GO, KEGG enrichment, molecular docking, and other technologies.o-methylisoxylitol are the main active components of bailing tablet, which acts synergistically on multiple targets to exert curative effects. According to the node characteristics of candidate compounds, a compound often corresponds to multiple targets, reflecting the multitarget treatment of vitiligo with bailing tablet. Furthermore, one compound can also come from a variety of herb, reflecting the advantages of compatibility of TCM. Quercetin belongs to flavonols. It was found that H2O2 could induce the expansion of endoplasmic reticulum and hinder the output of functional tyrosinase from the endoplasmic reticulum of melanocytes, thus resulting in the pathogenesis of vitiligo. Quercetin can reduce a series of oxidation reaction processes mediated by H2O2, and finally reduce the incidence of vitiligo. Baicalein is a flavonoid active substance extracted from Scutellaria baicalensis, and it can upregulate the expression of GPX4 gene, reduce the level of TFR1, inhibit the iron death of melanocytes, and enhance the antioxidant defense ability of human vitiligo melanocytes. Luteolin is a natural flavonoid with many pharmacological activities, such as anti-inflammatory, antiallergic, antioxidant, and so on. Inflammation and oxidation should play a major role in the pathogenesis of vitiligo. IL-8 is a major chemokine in inflammatory skin diseases. At the same time, the data proved that the expression of IL-8 gene in the skin of patients with vitiligo increases, thereby resulting in pathogenesis. With strong anti-inflammatory and antioxidant effects, luteolin can prevent the expression and release of IL-8 gene in melanocytes, which can reduce the incidence rate of vitiligo. Kaempferol is a flavonoid compound with anti-inflammatory and antioxidant effects, and it has been proved to be a melanopoietin. A variety of compounds contained in 11 herbs of bailing tablet exert their efficacy from different levels, such as cell, factor, protein synthesis, etc. These results are consistent with the prediction results of this study and could prove the reliability of the results.According to the “traditional Chinese medicine compound target” network, quercetin, baicalein, luteolin, kaempferol, arachidonic acid, calycosin, formononetin, prangenidin, phellopterin, and 7- Oxidative stress is the main pathogenesis of vitiligo. Although ESR1 could not increase the number of melanocytes, it could protect melanocytes from oxidative stress-induced cytotoxicity, thus cutting off the pathogenesis path of vitiligo.,19 Our results are similar to previous studies. KEGG enrichment analysis showed that bailing tablet mainly regulates immune inflammation, endocrine function, cell generation, apoptosis, autophagy, and other related pathways in the treatment of vitiligo. Among them, bailing tablet regulates the main pathways of melanogenesis, including p38/MAPK, CAMP/PKA, and PI3K/Akt. P38/MAPK can increase the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase, thereby inducing melanogenesis. Increased intracellular levels of CAMP can activate protein kinase (PKA), and CAMP can activate protein kinase (PKA) to phosphorylate CAMP-response element-binding protein and CAMP-response element binding protein-binding protein, thereby increasing the expression of MITF. PI3K/Akt signaling pathway inhibits melanogenesis by reducing the expression of tyrosinase, MITF and transient receptor potentical. In addition, bailing tablet also play a potential role in immune inflammation by mediating the differentiation of Th1, Th2, and Th17 and regulating the secretion of inflammatory factors such as IL-17, IL-6, tumor necrosis factor-α, and IL-1, which can reduce melanocyte injury. The expression of Th1 chemokine CXCL10 is significantly increased in patients with vitiligo, which is expected to become a new target for the treatment of vitiligo. In addition, JAK is also the main pathway of bailing tablet. There are literature studies on interferon (IFN)-γ after treatment, and it can lead to a concentration-dependent increase in chromatin condensation and nuclear fragmentation, which indicates that IFN- γ can directly induce mononuclear cell apoptosis. JAK-STAT is IFN- γ signal path, and some cytokines, such as leukocytes and IFNs, use the JAK-STAT pathway to transmit the signal from the cell membrane to the nucleus. Then the JAK protein was turned into an active protein, the phosphate STAT protein was darkened, and finally the nucleus was used to regulate the expression of related genes. Therefore, JAK inhibitors are often used to treat vitiligo. For example, patients with generalized vitiligo were treated with tofacitinib.According to the PPI network of bailing tablet, the degree value of TP53, ESR1, and other proteins is the largest, which may be an important target for its curative effect. The overexpression of TP53 (cell tumor antigen p53) can protect patients with vitiligo from photochemical damage and change the migration ability of melanocytes, so as to improve pigmentation in patients with vitiligo.According to the results of protein topology analysis and previous research support, we selected core proteins, including NTRK1, TP53, CUL3, ESR1, UBC, and epidermal growth factor receptor (EGFR), for molecular docking. The results revealed that the main compounds could bind well with core proteins. The docking score of ESR1 and NTRK1 is large, which further verifies that they may be the main protein targets of bailing tablet.Our study has some limitations. On the one hand, network pharmacology is a bioinformatics analysis based on databases. Due to the limitations of the databases, there may be deviations in the prediction target, and improving the database construction will enhance the credibility of the results. On the other hand, we analyzed the potential mechanisms of bailing tablet in the treatment of vitiligo, and the results need to be further verified by cell/animal experiments or clinical experiments.Bailing tablet plays a role in anti-inflammatory, antioxidation, and inhibition of melanocyte death. A compound can act on multiple targets related to the treatment of vitiligo, and each target can be related to multiple pathways. Clinically, bailing tablet is effective in the treatment of vitiligo, but the mechanism of action is not clear. This article explored the mechanism of bailing tablet in the treatment of vitiligo through multicomponent, multitarget, and multichannel through TCM network pharmacology and molecular docking technology. It highlights the scientificity and effectiveness of bailing tablet, provides guiding significance for clinicians in the treatment of vitiligo, and also provides new ideas for further exploring the mechanism of bailing tablet.Conceptualization and data curation: Jinming Li,Meng Yang.Funding acquisition and investigation: Yeqiang Song.Methodology and project administration: Jinming Li.


Both the cystic fibrosis transmembrane conductance regulator (CFTR) and the Ca2+-activated Cl– channel (CaCC) are Cl– channels involved in epithelial fluid secretion. Conjunctival fluid secretion could be increased by activating P2Y2R (an ATP receptor) in DED. However, the role of the CaCC and CFTR channels regulated by P2Y2R in eDED remains unclear. In this study, we established a rabbit eDED model using a controlled drying system. A Ussing chamber was used to perform a conjunctival short-circuit current induced by ATP to evaluate the reactivity of the ion channels to the ATP. Our results revealed that eDED accompanied by conjunctival fluid secretion impairment was caused by a P2Y2R dysfunction, which is related to CaCC-CFTR signaling in the conjunctiva epithelium. Notably, the coupling effect of the ATP-induced CaCC-CFTR activation and intracellular Ca2+ may represent a promising therapeutic target for treating eDED.Dry eye disease (DED) is a multifactorial disease with an incidence of approximately 50% worldwide. DED seriously affects quality of life and work. The prevalence of environmental DED (eDED) ranges from 35 to 48%. Conjunctival fluid secretion dysfunction may be one of the major causes of DED. Notably, the Cl More than one billion people suffer from dry eye disease (DED) worldwide. Severe DED can cause visual impairment, which severely affects the quality of life of patients ,2. DED i–) fluid. The outflow of the Cl– flux corresponded to the conjunctival fluid secretion [– channel that facilitates fluid secretion in the apical side of the conjunctiva [− concentration was found in patient tears with cystic fibrosis due to the CFTR mutation [The secretion of water from conjunctival epithelial cells was evaluated using chloride ion  [− flux in the airway epithelium [2+ ionophore) apically increased the Cl− flux by 58% in the rabbit conjunctiva epithelium [2+-activated Cl− channel (CaCC) was detected on the apical side of the conjunctiva [2R contributes to regulating the conjunctival fluid secretion via the combination of the CaCC and CFTR has not been elucidated, especially in eDED.ATP receptors exist in various types of epithelial cells, including the conjunctiva epithelium. P2Y rabbits ,11,12. Nn (Ca2+) , which hithelium . Moreoveithelium . A protejunctiva . These fjunctiva ,18. Whet2R signaling dysfunction is a key factor in the impairment of conjunctival secretion by the coactivation of the CaCC and CFTR. Our research revealed that a new molecular mechanism underlies the fluid secreted from the conjunctival epithelium, indicating a potential therapeutic target for treating eDED.In this study, the impairment and pathogenesis of conjunctival epithelium secretion in eDED was investigated using an eDED rabbit model. Our study demonstrated that the P2Yp < 0.001) (p < 0.001) (p < 0.001)  was established. In this study, the disruptions of the conjunctiva and cornea epithelium were stained with lissamine green and fluorescein, respectively. The staining score was positively correlated with the staining area. Compared with the control group, the lissamine green score increased from day 7 to day 14 in the dry eye group  a. Simila< 0.001) b. The fl< 0.001) c. Consis2+ and cAMP [2+ in eDED, we digested the eDED conjunctival epithelium and loaded it with an intracellular calcium fluorescence probe (Fluo-3/AM). The results showed that the intensity of the intracellular Ca2+ was decreased in the eDED conjunctival epithelium  (p < 0.05)   a. In add < 0.05) b. Subseqol group c. The ATroup (2R . The con < 0.05) a. These 2R and Isc variation, a P2Y2R inhibitor (suramin) was applied before the conjunctival ATP stimulation. The results showed that treatment with the P2Y2R inhibitor led to a decrease of 50% in the ATP-induced conjunctival Isc   (2R-siRNA eyedrop treatment (2R dysfunction.To elucidate the relationship between the P2Y < 0.01) b. The AT < 0.05) c. The rareatment d. These – flux and HCO3− flux were secreted from the Cl– channel of the cell membrane surface [− or HCO3− was used to determine whether the ATP-induced conjunctival Isc variation is related to the exchange of Cl– or/and HCO3–. Compared with the normal buffer solution, the ATP-induced conjunctival Isc response decreased by approximately 50% in the Cl–-free buffer     a,b,e and< 0.001) b,c,e. Mo< 0.001) a,d,e. Th− channel that is essential for conjunctival fluid secretion [89) were applied before the conjunctival ATP simulation. The CFTR inhibitor reduced the second peak of the conjunctival Isc without affecting the first peak    (The CFTR is a cAMP-dependent Clecretion . To dete < 0.01) a,b,f. A < 0.001) c,f. Furt< 0.001) d,f. Thes– channel, CaCC, which is a Ca2+-activated Cl– channel on the cell membrane of the conjunctiva epithelium [p < 0.05; second peak: 22.16 ± 2.40 vs. 10.95 ± 4.11, n = 5–14, p < 0.05)  e,f. Thes2+ in the ATP-evoked conjunctival Isc, a PLC inhibitor (U73122), an IP3R inhibitor (2-APB), and a Ca2+ chelator (BAPTA/AM) were applied before the ATP simulation. The ATP-induced conjunctival Isc in the PLC inhibitor group decreased to one-half of that in the vehicle group  (p < 0.001)   a,b,e. A < 0.001) a,c,e. Th in eDED d as wellitor U731, an IP3R < 0.05) a,d,e. Th2R dysfunction combined with the decreased intracellular cAMP and cytosolic Ca2+ in the conjunctival epithelium was demonstrated in an eDED rabbit model. The P2Y2R dysfunction caused a conjunctival fluid secretion impairment, which was derived from the ATP through the CaCC-CFTR signaling. The release of the intracellular Ca2+ plays a core cross-pivotal role in the CaCC-CFTR signaling and is indispensable for the ATP-induced conjunctival fluid secretion. Therefore, the impairment of the conjunctival fluid secretion via the CaCC-CFTR pathway dysfunction could be a key mechanism in eDED.In this study, P2Y− from the conjunctival basal side to the apical side via an exchange with the sodium ions was demonstrated to account for approximately 60% of the Isc response [3− and other anions could occupy more than one-third of the ATP-induced Isc response. A similar fluid secretion was also detected in the cortical collecting duct cells [The previous studies have shown that fluid in the conjunctiva epithelium is transported from the stromal  side to the mucosal  side. Conjunctival fluid secretion is dependent on the outflow of the anion flux . There aresponse ,26. Therct cells  and the ct cells .2+ are elevated after the P2Y receptor activation [2R is interrelated with the increase in the PLC and the transient increase in the intracellular Ca2+ concentration [2+ may regulate the conjunctival fluid secretion. Moreover, a release of the intracellular Ca2+ by another way, but not the PLC pathway, could have the same impact [2R is a key factor in the conjunctival fluid secretion. However, the concentration of the ATP in tears increased in dry eye patients [2R dysfunction, was believed to cause increased tear ATP. The results of the present study showed that the P2Y2R dysfunction with decreased intracellular cAMP and Ca2+ existed in the conjunctiva of eDED, suggesting that P2Y2R plays a key role in eDED. In contrast, silencing P2Y2R led to a dry eye condition, and the inhibition of P2Y2R effectively suppressed the ATP-induced conjunctival Isc.To date, the activation of the P2Y receptors has been considered to stimulate the fluid secretion across the conjunctiva . The levtivation ,20. The ntration , suggeste impact . These rpatients . The con− channel existing in both the corneal and conjunctival epithelium, which increased the basal tear volume after activation [The CFTR is a cAMP-dependent Cltivation . The pre− channel, exists in the apical surface of the conjunctiva epithelium [2+ dependent Cl– channel that transports the fluid secretion from the airway epithelium [2+ activated a transient opening of the CaCC and then a persistent CFTR channel for the Cl− secretion from the conjunctival epithelial cells. This study is the first in the literature to verify the combination of the CaCC signaling and CFTR in the Cl– outflow across the conjunctival epithelium.In this study, the CaCC inhibitor decreased the ATP-induced conjunctival Isc, indicating that the CaCC signaling may be the main contributor to the ATP-induced conjunctival fluid secretion. The CaCC, another Clithelium . The CaCithelium . Our res2+ in the conjunctiva epithelium was decreased in eDED and did not respond to the ATP activation. The level of the intracellular Ca2+ was shown to be closely related to the function of the epithelial secretion, such as in the airway epithelium [2+ in the conjunctiva epithelium can be regulated via different mechanisms. The activation of the transient receptor potential melastatin 8 channel increased the intracellular Ca2+ and then conducted sense information to the higher brain center [2R dysfunction was found in the conjunctiva epithelium in eDED, which may be an intrinsic cause of the decreased intracellular Ca2+. Except for P2Y2R, the imbalance in the intracellular Ca2+ could be affected by endoplasmic reticulum stress and a mitochondrial matrix Ca2+ overload, which triggered cell apoptosis [2+ in eDED.Our results showed that the content of the intracellular Caithelium , caudal ithelium , and conithelium . The intn center . In thispoptosis ,35. EndoMale New Zealand white rabbits weighing 2.0–2.5 kg  were chosen for the study. The rabbits were quarantined and acclimatized for one week before the experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Zhongshan Ophthalmic Center at Sun Yat-sen University and complied with all relevant ethical regulations for animal testing and research.The rabbit eDED model was established using a controlled drying system (CDS). A detailed system for dry eye models was published in our previous report . In brie2R were applied topically four times per day to the ocular surfaces of the rabbits. The nucleotide sequence of the siRNA target site chosen to silence the expression of the rabbit P2Y2R-coding sequence (GenBank EU886321) was as follows: 5′-AAC CTG TAC TGC AGC ATC CTC-3′ [Small interfering RNA (siRNA) sequences for silencing P2YC CTC-3′ ,39. The 2. The side of the epithelium was placed in a fixed direction facing the positive electrode. Then, the conjunctiva was bathed on both sides with bicarbonate Ringer’s solution composed of the following (in mM): 116.4 NaCl, 5.4 KCl, 5.4 NaHCO3, 0.78 NaH2PO4, 1.8 CaCl2, 0.81 MgCl2, 5.55 D-glucose, and 10 HEPES. It was then gassed with 95% O2/5% CO2 at 37 °C to maintain a pH of 7.4. The epithelium exhibited a basal transepithelial potential difference, which was measured by Ag/AgCl electrodes with KCl/Agar bridges connected to a voltage-clamp amplifier . The transepithelial potential difference in the conjunctiva epithelial layer was clamped at 0 mV for the Isc measurement.The Isc measurement was performed following the modified procedure described in our previous reports . The supThe change in Isc was defined as the difference between the value at baseline and that at a peak following compound addition, displayed via a signal collection and analysis system . When the current changed to the 1 mV pulse, it was used to estimate transepithelial resistance according to Ohm’s law . Ten min–-free BRS, NaCl, KCl, and CaCl2 were replaced by the respective salts of gluconate. In the HCO3–-free solution, HCO3− was substituted by 10 mM HEPES and bathed with 100% O2 to maintain pH at 7.4. In the Cl–- and HCO3–-free solution, the components were the same as in the Cl−-free solution, except that NaHCO3 was replaced by 20 mM Na-gluconate and 10 mM HEPES. Inhibitors or a vehicle  were added to the buffer medium for 20 min before ATP was applied to the apical side. The dosages of inhibitors were as follows: 100 uM U73122 , a phospholipase C (PLC) inhibitor; 100 uM 2-APB , an inositol  trisphosphate (IP3) receptor (IP3R) inhibitor; 100 uM BAPTA/AM , a Ca2+ chelator; 40 uM T16Ainh-A01 , a specific CaCC inhibitor; and 40 uM CFTR (inh)-172 , a specific CFTR inhibitor; 10uM H89 , a specific cAMP-dependent protein kinase (PKA) inhibitor; and 30 uM MDL12330A , an adenylate cyclase (AC) inhibitor. Different inhibitors were individually used in the conjunctival samples.In the ClThe concentration of intracellular cAMP in the conjunctival epithelium was measured using a cAMP assays kit  according to the manufacturer’s protocol. Optical density readings were measured using a SynergyTM H1 multifunctional microplate reader . Concentrations were determined using a standard curve as a reference.2+ was measured using the fluorescence dye Fluo-3/AM according to a modified procedure described in a previous report [Intracellular Cas report . The conp was less than 0.05.All statistical analyses were performed using SPSS version 22.0. All results were expressed as mean ± SEM. Statistical significances were determined using a Student’s t-test (two groups), and a repeated measures ANOVA, followed by a Least significant difference post hoc test (groups with repeated measurements). The differences between the groups were considered statistically significant if 2+-activated CaCC-CFTR pathway plays a key role in conjunctival secretion. The impairment of the P2Y2R-initiated CaCC-CFTR may be a key mechanism of the conjunctival secretion dysfunction in eDED.The results of this study indicate that the Ca


Our simulation study demonstrates that the power of long-KMR exceeded that of another KMR method previously developed to analyze longitudinal data, while maintaining (slightly conservatively) the type I error. The network kernel improved the performance of long-KMR compared to the linear kernel. Considering different pathway densities, the power of the network kernel decreased with increasing pathway density. We applied long-KMR to cognitive data on executive function  from the PsyCourse Study and 17 candidate pathways selected from Reactome. We identified seven nominally significant pathways.A popular approach to reduce the high dimensionality resulting from genome-wide association studies is to analyze a whole pathway in a single test for association with a phenotype. Kernel machine regression (KMR) is a highly flexible pathway analysis approach. Initially, KMR was developed to analyze a simple phenotype with just one measurement per individual. Recently, however, the investigation into the influence of genomic factors in the development of disease-related phenotypes across time (trajectories) has gained in importance. Thus, novel statistical approaches for KMR analyzing longitudinal data, i.e. several measurements at specific time points per individual are required. For longitudinal pathway analysis, we extend KMR to long-KMR using the estimation equivalence of KMR and linear mixed models. We include additional random effects to correct for the dependence structure. Moreover, within long-KMR we created a topology-based pathway analysis by combining this approach with a kernel including network information of the pathway. Most importantly, long-KMR not only allows for the investigation of the main genetic effect adjusting for time dependencies within an individual, but it also allows to test for the association of the pathway with the longitudinal course of the phenotype in the form of testing the genetic time-interaction effect. The approach is implemented as an R package,  Pathway analyses or gene-set analyses are association studies, which test whole gene sets or pathways for association with a phenotype of interest . In contIn this paper, we focus on kernel machine regression (KMR) , a machiLongitudinal studies assess multiple, thus correlated, measurements over time for each single individual . They enIn the genetic context, these LMMs can be applied to perform longitudinal GWASs . Using tThe aim of this work is to develop a longitudinal pathway analysis to test for the association between genetic factors and the longitudinal phenotype applying KMR and simultaneously allowing integrating network information. To be able to analyze longitudinal data, we extended KMR to long-KMR. Other authors have also studied longitudinal data  and creaIn a simulation study, we assessed the properties of long-KMR regarding several aspects. We considered longitudinal studies with two and four measurement points. We compared the performance of long-KMR when applying a linear kernel or a network kernel. We also studied the influence of the pathway topology on the performance of the network kernel with a focus on the density of the pathway.Finally, as a real-world application, we use long-KMR on the data from our previous longitudinal GWASs  on execuIn summary, in this paper we first present the theoretical aspects of long-KMR and the network kernel. We then describe the simulation approach used to evaluate our method, and, lastly, provide a real-world example of long-KMR.In this section, we introduce the KMR analysis and its extension to analyze longitudinal data. We describe our simulation approach to investigate the type I error rate and power. Lastly, we present an application of long-KMR as example and give details on the PsyCourse Study data and the pathways used.Let us assume  product . The repuct  has bees . Here we set α to equal 5%, 1%, 0.5%, and 0.1%, respectively. In the scenarios in which we simulated genetic effects, we determined the power as the proportion of simulations with a p-value <5% threshold. In total, we compared the power for three different genetic effect models. We simulated two single-effect models containing either a main genetic effect or a time-interaction effect. We also created a more complex model, the joint model, which comprises a main genetic effect and a time-interaction effect. The joint model was only studied in a limited number of scenarios, as portrayed in We studied type I error rates and power in different scenarios to assess the performance of long-KMR for different genetic effects and the network kernel. The type I error rate is defined as the proportion of simulations that have a igraph package  of a pathway. This density is a graph-theoretical characteristic defined as the ratio of the number of present connections divided by the maximum number of possible connections in a pathway .For the null model corresponding to the null hypothesis of no genetic effects, we simulated the quantitative phenotypes according to the following LMM for an individual larly to . For thelarly to .For the power simulations, we added genetic effects to our null model to simulate the phenotypes. All models comprised three causal “pseudo-” genes each with three causal SNPs . The effect sizes p-value, which we combined with the Fisher’s method  . TMT-B ihttps://www.cog-genomics.org/plink/) to compute the ancestry principal components.Genotyping was performed with the Illumina Infinium Global Screening Array-24 Kit (version 3.0 or version 1.0) and the imputation took place on the Michigan imputation server  with theigraph package (https://reactome.org/content/detail/R-HSA-1236394) as foundation pathway for our simulation study. This pathway was selected for its moderate size of 19 genes and because it only contains one CHEBI. The network consists of only one graph component, also denoted as connected . Most importantly, the pathway has an intermediate density of 0.46, which was a good basis for further artificial pathways we generated with high and low densities. “Signaling by ERBB4” is connected to schizophrenia . We included the following fixed effects in the model: sex, age at first measurement point, diagnostic group , time, and the top five ancestry principal components. A random intercept and a random slope for the time effect were also added. We tested each pathway for a potential main genetic and a time-interaction effect. The linear kernel (LIN), the unadjusted network (NET), and the size-adjusted network kernel (ANET) were applied. We assigned a SNP to a gene of a pathway based on its genomic location with a mapping window of rding to . We compapproach . We detehttps://gitlab.gwdg.de/bernadette.wendel/kalpra. In addition to the linear and network kernels, a quadratic kernel is also available. The pathway information can be directly downloaded and transformed into an annotation and adjacency matrix. The computational aspects for some example analyses are provided in We performed all analyses with R , which wp-value < α  in the null simulations without genetic effects. The type I error rates were maintained overall at the different α thresholds for the models in our simulation scenarios. We did detect individual type I error rates only slightly exceeding the respective significance levels, e.g., 5% and 1%, for three models . However, all the values lie in the range of expected random variations . KMR-NET-d0.8-m2 presented the largest increase in type I error of 5.09% at the significance level of 5%. p-values of the KMgene analysis revealed an inflation of the error rates. The error rates for the analyses of the missing aspect for the different network kernel were also maintained  compared to 33%–36% (main genetic effect)). Overall, the time-interaction effect yielded a higher power for the four-measurement models, especially for the smaller effect sizes 0.04 (For the two single-genetic-effect models (main genetic and time-interaction model), the power comparison of the two-measurement models revealed that the LMM had the highest power independent of effect size and for either kernel. ANCOVA had the lowest power for the two-measurement models in comparison. zes 0.04  and 0.06zes 0.04 . An addizes 0.04 . In the zes 0.04 . As dispβ = 0.04, 0.06, and 0.08). For example, for KMR-LIN-m4 testing the main genetic effect, the power increased from 36% (β = 0.04) to 78% . This effect increased with increasing effect size β.The analyses performed with different percentages of missing data revealed similar features for the single-effect models, with a general decrease of power compared to the analysis of a complete phenotype data set . In geneIn our real-world data sample, we analyzed 1,518 individuals with at least one TMT-B measurement including 591 “affective,” 533 “psychotic,” and 394 mentally healthy individuals. The mean age at the first measurement point was 41 years [sd: 13.8] 48% of the samples were female  for either applied kernel . However, we identified seven pathways in total as achieving a p-value <0.05, which are represented in bold in p-value of all pathways (0.0089). To compare the different kernels, we ranked all pathways according to their p-values. p-value ranking, the “synaptic adhesion-like molecules” pathway stood out as the one with smallest p-value (rank 1) in all analyses.Thirteen of the 17 tested pathways overlapped at least with one other pathway in at least one gene. The four independent  were “ion channel transport,” “EPH-ephrin signaling,” “receptor-type tyrosine-protein phosphatases,” and “regulation of MECP2 expression and activity” . We did Here we present long-KMR, a topology-based pathway analysis method for longitudinal data, which applies kernel machine regression. The methodological basis of long-KMR is presented. To create long-KMR the connection of KMR and LMM are exploited. In addition, we use the network kernel  integratAs expected, the power of long-KMR increases with growing number of measurement points, in particular for the time-interaction effect. This can be traced back to the information that is added to the model at each measurement point, increasing the probability of detecting an effect. Thus, we also identified a larger power loss when analyzing the time-interaction effect with incomplete phenotype data (missing measurements).The performance of long-KMR improves further when we apply the network kernel instead of the linear kernel, in particular in the single-effect models. We observe that the network kernel has at least the same power as the linear kernel. The power benefit of the network kernel is more pronounced when testing in the presence of smaller genetic effect sizes. For larger genetic effect sizes the power is already extremely high  still has the highest power but only slightly higher when compared to the linear kernel (approx. 72%–73%). The network kernels with densities 0.5 and 0.8 have surprisingly low power compared to the linear kernel. This phenomenon is perhaps due to the simulation of the genetic effect as purely linear effect and enhanced by the application of two different kernel functions in one model. We simulate the genetic effects in a linear fashion and thus we observe the performance of the network kernel in worst-case scenarios. Nevertheless, the network kernel improved long-KMR slightly when the pathway density is not too high. In general, long-KMR is preferable except when testing a very dense pathway. The latter should be acknowledged and considered when interpreting the results of long-KMR under a specific kernel.It should also be taken into account that a possible misspecification of a pathway, for example, in the form of wrongly described gene connections leads to an inaccurate pathway topology and pathway characteristics, e.g., density. This can lead to power changes in the analysis. Thus, one of the greatest challenges to topology-based pathway analyses remains the possible inaccuracy and perhaps incompleteness of the studied pathways. Here, future work is required to minimize possible misclassifications. In the future, it would be also worthwhile analyzing other pathway characteristics, e.g., the betweenness centrality or diameter of the pathway, and their influence on power of the long-KMR with the network kernel. However, these aspects should also be considered beforehand in one-measurement settings in order to determine any indication of the performance being affected and thus keep the computational costs associated with the analysis of an extensive longitudinal scenario down to an acceptable level. Additionally, more complex simulation models could be considered, including e.g., genetic effect models in which causal SNP effects interact with each other and the causal SNPs vary between main and interaction effects. Here it is expected that these scenarios are even more advantageous for the network kernel. However, this exceeds the scope of this communication.When comparing the different two-measurement models either for the main effect or for the time-interaction effect, long-KMR has the higher power and is the preferred option, in spite of its longer computation time, in particular when using the network kernel. As expected ANCOVA has lower power. Note that the ANCOVA model only uses the second measurement point as dependent variable  and losep-value combination approach according to Stouffer . Thus, our approach represents the suitable choice when analyzing a whole pathway. KMgene remains a solid approach when analyzing single genes.For the four-measurement models the comparison with KMgene  on pathwStouffer , yieldinp-value <0.1, i.e., “synaptic adhesion-like molecules,” “signaling by ERBB4,” “long-term potentiation,” and “NCAM signaling for neurite growth.” The first three pathways contain the gene DLG4. This synaptic gene encodes for the density protein 95 (PSD95) and plays a critical role in the activity regulation of NMDA (N-methyl-d-aspartate) receptors in schizophrenic patients . Ion channels are implicated in influencing the susceptibility to or the pathogenesis of psychiatric diseases (Of the seven pathways nominally significant, “ion channel transport” was the only pathway to prove significant for the time-interaction effect and when modelled with the linear kernel. This pathway had the lowest diseases , and arehttps://gitlab.gwdg.de/bernadette.wendel/kalpra.Overall, we demonstrated that our longitudinal topology-based pathway analysis displays a power gain and a great flexibility to model pathways and genetic effects. Our approach enables the choice between the popular linear kernel and a network kernel that integrates pathway topology information. The latter demonstrated superiority depending on the density of the pathway of interest. The approach is implemented as the R package kalpra, which is available at


Non-specific orbital inflammation (NSOI) and IgG4-related orbital disease (IgG4-ROD) are currently treated with non-specific immunosuppressive agents based on non-randomized, uncontrolled studies. Therefore, relapses and prolongated courses are common and remain challenging. For a more specific therapy, a better understanding of the underlying pathophysiology is crucial. Therefore, we aimed to analyze signaling pathways to expand the knowledge on the pathophysiology and possibly identify specific targets in the future, as occurred recently in Graves’ orbitopathy with the IGF-1 receptor. Furthermore, we analyzed potential mechanisms for the described potential progression to orbital MALT (mucosa-associated lymphoid tissue) lymphoma. The investigation cohort for this screening study comprised of 12 patients with either typical NSOI (n = 6), IgG4-ROD or MALT lymphoma (n = 3 each). Mean age was 56.4 ± 17 years. MALT samples, in contrast with IgG4-ROD and NSOI, showed overall upregulation for extracellular matrix receptor interaction (ECM) and adipocytokine signaling. Investigating signaling compounds for MALT samples, differentially expressed genes were re-identified as targets with relevant expression. Even though pathway analysis showed differentially altered products when comparing IgG4-ROD with MALT, main conductors of differentiation in B- and T-cell signaling were commonly altered when observing the microenvironment of examined tissues. Our data reveal the characteristic differences and similarities in genetic-expression-based pathway profiles between MALT lymphoma, IgG4-ROD and NSOI, which may be useful for elucidating the associated pathogenic mechanisms and developing specific treatments for these orbital diseases. The term orbital inflammatory disease (OID) encompasses a wide range of different orbital diseases. The most common of these is the recently discovered Graves’ orbitopathy (GO), which is probably why our understanding of its pathophysiology has highly advanced in recent years ,4,5,6,7.This study is based on our previously published study . In the RNA extraction of the 12 routinely processed formalin-fixed and paraffin-embedded biopsy specimens was performed as previously described . In shorBased on current literature, we selected multiple genes involved in tumor- and inflammation-associated pathways. Gene expression patterns were screened using the NanoString nCounter platform for digital gene expression analysis with the appurtenant PanCancer Progression Profiling panel, consisting of 770 genes and the Immunology V2 Profiling panel consisting of 594 genes mediating immune response as well as 30 reference genes see . Hybridit-test of negative controls and target specific counts in all samples to identify genes not relevantly expressed (p < 0.05) [Count data acquired by NanoString analysis were normalized and analyzed using the R statistical programming environment . Considering the counts obtained for positive control probe sets, raw NanoString counts for each gene were subjected to a technical factorial normalization, carried out by subtracting the mean counts plus two-times the standard deviation from the CodeSet inherent negative controls. Subsequently, a biological normalization using the included RNA reference genes was performed. In addition, background noise was excluded by utilization of one-sided Wilks  < 0.05) .t-test (parametric). For comparison of ordinal variables and factors with more than two groups, either the Kruskal–Wallis test (non-parametric) or ANOVA (parametric) was used to detect group differences. Correlations between metrics were tested applying Spearman’s rank correlation test as well as Pearson’s product-moment correlation testing for linearity. Basic quality control of run data was performed by mean vs. variance plotting in order to find outliers in target or sample level. True differences were calculated by correlation matrices analysis. Quality control of run data was first performed in basic by mean vs. variance plotting to find outliers in target or sample level. True differences and clusters on both target and sample level were calculated by correlation matrices analysis. Pathway analysis is based on the KEGG database (Kyoto Encyclopaedia of Genes and Genomes) and was performed using the “pathview” package in R. Differences were specified by log2-fold changes between means (parametric) or medians (non-parametric) of compared groups. Significant pathway associations were identified by gene set enrichment analysis using the WEB-based GEne SeT AnaLysis Toolkit  [p < 0.05).Statistical and graphical analyses were also performed within the R statistical programming environment (v. 4.0.3). Prior to exploratory data analysis, the Shapiro–Wilks test was applied to test for normal distribution of each data set for ordinal and metric variables. Resulting dichotomous variables underwent either the Wilcoxon/Mann–Whitney rank sum test (non-parametric) or two-sided Student’s Gestalt) ,33,34,35p-values were adjusted by using the false discovery rate (FDR). The level of statistical significance was defined as p ≤ 0.05 after adjustment.Due to the multiple statistical tests the For each analyzed group, a gene set enrichment analysis (GSEA) was performed. GSEA utilizes molecular interaction networks outlined by the Kyoto Encyclopedia of Gene and Genomes (KEGG) to map out increased gene expression in a specific molecular pathway, depending on a response variable (entity) . Therebyp-value: 0.001), Chagas disease , Staphylococcus aureus infection , phospholipase D signaling pathway  and the neurotrophin signaling pathway pertussis , Fc epsilon RI signaling pathway , platelet activation .apoptosis , ECM (extracellular matrix)–receptor interaction , complement and coagulation cascades  and focal adhesion , amoebiasis Focal surface regulatory pathways: protein digestion and absorption , AGE-RAGE signaling pathway in diabetic complications , small-cell lung cancer , relaxin signaling pathway Effector pathways with systematic impact: complement and coagulation cascades , T-cell receptor signaling pathway , neurotrophin signaling pathway , thyroid hormone signaling pathway Focal surface regulatory pathways: B-cell receptor signaling pathway , hepatocellular carcinoma  and FoxO signaling pathway Effector pathways with systematic impact: cell cycle , complement and coagulation cascades , ECM–receptor interaction , Salmonella infection  as well as cytokine–cytokine receptor interaction NOD-like receptor signaling pathway  herpes simplex infection , and rheumatoid arthritis Effector pathways with systematic impact: hematopoietic cell lineage , B-cell receptor signaling pathway , T-cell receptor signaling pathway , neurotrophin signaling pathway , platelet activation , insulin signaling pathway , phospholipase D signaling pathway , as well as the thyroid hormone signaling pathway Fc gamma R-mediated phagocytosis  were previously reported between each of our groups [IGF1  when compared to NSOIs and IgG4-ROD. For IgG4-ROD vs. all, expression was not significantly altered ; looking at our data (IGFBP) were also significantly differentially expressed as IGFBP4  and IGFBP7   .Observing the extracellular matrix (ECM)–receptor interaction compounds, IgG4-ROD samples had transcripts responsible for ECM adhesion altogether with their integrin domains downregulated compared to MALT lymphoma samples; namely, collagen, laminin, thrombospondin (THBS), α1, α 5, α 6, α 7 and β4 (ITGB4). Syndecan showed upregulation for both IgG4-ROD and MALT .TNFR1, mTOR, AKT1 , SOCS3  and STAT3 , followed by NSOI showing a slight upregulation of mTOR and AKT compared to overall downregulation of compounds in IgG4-ROD  biomarker genes. After receiving multiple questions about the possible origin and mechanisms resulting in these differentially expressed genes, we performed a gene set enrichment analysis for this study to provide a more detailed insight into the pathophysiology.mTOR, AKT, SOCS3 and STAT3 that also project insulin resistance effects through various downstream ligands. In contrast to the reduced expression in IgG4-ROD and NSOI samples, we see elevated expression levels in MALT samples. MALT samples showed diverging results when compared to all other OID, which was expected due to the nature of the disease from a biological point of view, though from a clinical approach the differentiation is not this easy [Rosenbaum et al. recently published a study explaining IGF1-R and PPARγ enrichment in multiple OID ), but not IgG4-ROD. Validation of our results is supported by the expression levels in the adipocytokine signaling pathway of NSOI when compared to the pathway analysis . Mainly his easy . A main his easy . CompariAdipocytes themselves have been described as contributors to fibrosis many times before. Adipokines such leptin and adiponectin have been implicated to hepatic fibrosis , orbitalThe innate immune system is the first line of defense against microbial invasion, relying on pattern recognition receptors to recognize external pathogenic microorganisms and then remove them . The nucInformation about expression levels of NOD-Like receptor signaling pathway in our MALT samples has been added as Interestingly both groups showed a decreased enrichment score for the B-cell receptor signaling pathway, T-cell receptor signaling pathway and Neurotrophin signaling pathway.Chlamydia spp. could be a co-factor in some cases for this progression as previous studies observed, similar to the common oncogenesis of gastric MALT lymphoma following chronic gastritis with Helicobacter pylori infection [Typically, a B-cell is defined and created by the productive rearrangement of immunoglobulin heavy (IgH) and light (IgL) chain genes, leading to expression of a B-Cell receptor (BCR). Each B-cell is encoded by a unique molecular fingerprint that results of unique sequences of it IgH and IgL hypervariable regions (HVRs) . In gastnfection . Others nfection ,57,58 inAfter recognition of IgG4-ROD as a unique disease entity, the humoral immune response received much attention, principally because of the hypergammaglobulinemia and the prominent IgG4-positive cells in both affected tissue and patient blood samples, disappearing upon corticosteroid treatment . FurtherPreviously mentioned B-cell expansion taking place in secondary lymphatic tissue via iterative rounds of mutation and positive selection within germinal centers, is a T-cell-dependent process. In vitro studies have shown that T follicular helper cells (Tfh) associated with different Th subsets differentially shape the quality of human humoral immunity. A study investigated the role of CXCR5+ CD4+ T-cells circulating through the human blood . Their rLimitation of this study include its relatively small number of patients that are collected from only one institute and its retrospective design. This might have resulted in selection and confounding bias. Furthermore, the heterogeneous histopathological findings and orbital locations might mask gene expression differences that are only present in specific subtypes of the entities, especially in NSOI. However, since the main goal of this study was to differentiate pathophysiological aspects between NSOI, IgG4 and MALT, this broader analysis revealed the most prominent pathways for each entity. Further analysis of IgG4-ROD and GO should be conducted via elementary methods, such as immunoassays and in cell cultures, before serious therapeutic assumptions can be drawn. We see a validation of our results comparing IgG4-ROD and NSOI pathway analysis with other study groups. A previous analysis of OID with neoplastic disorders in this form has only been conducted in our previously published study, which makes a literature review for validation purposes difficult. However, looking at compound expression of our preformed GSEA, we can see a validation of molecular mechanisms as expected.In our study, we show that though NSOI, IgG4-ROD and orbital MALT lymphoma share clinical symptoms, the enriched pathways are significantly different. Especially, MALT lymphoma showed completely different pathway patterns, i.e., elevated ECM–receptor interaction and adipocytokine pathways in contrast to downregulations in IgG4-ROD and NSOI samples. In contrast, NSOI was prominent in the activation of innate immune pathways; both IgG4-ROD and MALT showed significantly decreased enrichment in definitive cell differentiation conductors: B- and T-cell signaling pathways. These common pathways support the hypothesis that chronic inflammation of IgG4-ROD could lead to orbital MALT lymphoma by malignant transformation of lymphocytes.


Zanthoxylum bungeanum in the treatment of diabetes mellitus (DM) using network pharmacology.  The aim of the study was to explore the potential mechanism of Zanthoxylum bungeanum, and the genes related to diabetes mellitus were obtained from the genecards database. Import the data into the Venny 2.1.0 platform for intersection analysis to obtain the Zanthoxylum bungeanum-DM-gene dataset. The protein-protein interaction (PPI) analysis of Zanthoxylum bungeanum-DM gene was performed using the String data platform, and the visualization and network topology analysis were performed using Cytoscape 3.8.2. The KEGG pathway enrichment and biological process of GO enrichment analysis were carried out using the David platform. The active ingredients and key targets of Zanthoxylum bungeanum were molecularly docked to verify their biological activities by using Discovery Studio 2019 software. Zanthoxylum bungeanum was extracted and isolated by ethanol and dichloromethane. HepG2 cells were cultured, and cell viability assay was utilized to choose the suitable concentration of Zanthoxylum bungeanum extract (ZBE). The western blot assay was used for measuring the expression of AKT1, IL6, HSP90AA1, FOS, and JUN proteins in HepG2 cells.  The DrugBank database and TCMSP platform were used to search for the main chemical components and their targets of Zanthoxylum bungeanum are kokusaginin, skimmianin, diosmetin, beta-sitosterol, and quercetin, respectively. The main targets for its antidiabetic effect are AKT1, IL6, HSP90AA1, FOS, and JUN, respectively. GO enrichment analysis revealed that the biological process of Zanthoxylum bungeanum and DM is related to a positive regulation of gene expression, positive regulation of transcription, positive regulation of transcription from RNA polymerase II promoter, response to drug, positive regulation of apoptotic process, and positive regulation of cell proliferation, etc. KEGG enrichment analysis revealed that common biological pathways mainly including the phospholipase D signaling pathway, MAPK signaling pathway, beta-alanine metabolism, estrogen signaling pathway, PPAR signaling pathway, and TNF signaling pathway. Molecular docking results showed that AKT1 with beta-sitosterol and quercetin, IL-6 with diosmetin and skimmianin, HSP90AA1 with diosmetin and quercetin, FOS with beta-sitosterol and quercetin, and JUN with beta-sitosterol and diosmetin have relatively strong binding activity, respectively. Experiment verification results showed that DM could be significantly improved by downregulating the expression of AKT1, IL6, HSP90AA1, FOS, and JUN proteins after being treated at concentrations of 20 μmol/L and 40 μmol/L of ZBE.  A total of 5 main compounds, 339 targets, and 16656 disease genes were obtained and retrieved, respectively. A total of 187 common genes were screened, and 20 core genes were finally obtained after further screening. The antidiabetic active ingredients of Zanthoxylum bungeanum mainly including kokusaginin, skimmianin, diosmetin, beta-sitosterol, and quercetin. The therapeutic effect of Zanthoxylum bungeanum on DM may be achieved by downregulating core target genes including AKT1, IL6, HSP90AA1, FOS, and JUN, respectively. Zanthoxylum bungeanum is an effective drug in treatment of DM related to the above targets. The active components of  Diabetes mellitus (DM) is one of the most serious endocrine disorders and noncommunicable diseases that threaten human health worldwide, mainly manifested as carbohydrate, fat, and protein metabolism disorders; the prevalence rate is increasing globally . AccordiZanthoxylum bungeanum is widely distributed in China, mainly in Sichuan, Guizhou, Gansu, Shaanxi, Chongqing, and other provinces. Zanthoxylum bungeanum is a traditional Chinese medicine (TCM) with a long history of edible and medicinal use [Zanthoxylum bungeanum is warm in nature, pungent in taste, and returns to the spleen, stomach, and kidney meridians, and has the effects of warming and relieving pain, killing insects and relieving itching. The medicinal parts are the dried and ripe peel of the Rutaceae plant green pepper (Zanthoxylum schinifolium Sieb.et Zucc.) or Chinese Zanthoxylum bungeanum (Zantohxylum bungeanum Maxin.) [Zanthoxylum bungeanum is rich in chemical components, mainly including volatile oils, alkaloids, amides, flavonoids, lignin, coumarin, triterpenoids, and other compounds [Zanthoxylum bungeanum are manifested in analgesia, anti-inflammatory, antioxidant, antibacterial, and antiobesity [Zanthoxylum bungeanum exhibits the antidiabetic effect [inal use . It is r Maxin.) . The peeompounds . The phaiobesity . In recec effect , 11.Zanthoxylum bungeanum, but few studies have combined the chemical constituents and network pharmacology of Zanthoxylum bungeanum. With the development of bioinformatics and network pharmacology, technologies and methods based on biological data integration provide a convenient way to clarify the complex mechanism of action of TCM [Zanthoxylum bungeanum in the treatment of DM. With the help of various public databases to obtain the effective active components of Zanthoxylum bungeanum and related antidiabetic targets, predict the antidiabetic targets of Zanthoxylum bungeanum, and conduct enrichment analysis for the relevant targets. We use Discovery Studio 2019 software for molecular docking to study the binding ability between receptors and small drug molecules and provide a reference for its in-depth research and clinical application. This study combined network pharmacology and molecular docking verification to explore the antidiabetic mechanism of Zanthoxylum bungeanum.Due to the complex components of TCM that takes a lot of manpower, material resources and time to study the molecular mechanism of TCM through animal experiments or cell experiments. In recent years, many scholars have studied the chemical constituents and pharmacological effects of n of TCM . In the Zanthoxylum bungeanum were collected through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), the threshold of oral bioavailability (OB) was ≥30%, and molecular weight (WM) was <500; the compounds were screened based on the drug-likeness (DL) threshold ≥ 0.18. After screening the main chemical components of Zanthoxylum bungeanum, the targets of active ingredients of Zanthoxylum bungeanum were screened out through the TCMSP analysis platform and DrugBank database.All the chemical components of https://www.genecards.org/) and DisGeNET database (https://www.disgenet.org/) to search with “diabetes” OR “ diabetes mellitus” OR “DM” to obtain diabetes disease targets, respectively. The Venny 2.1.0 online mapping tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to match the targets related to the active components of Zanthoxylum bungeanum obtained in Zanthoxylum bungeanum.The GeneCards database provides comprehensive, user-friendly information on all annotated and predicted human genes. The DisGeNET database containing one of the largest publicly available collections of genes and variants associated with human diseases. Using the human GeneCards database  was used to search for known and predicted PPI. Input the common targets of Zanthoxylum bungeanum and DM into the String database, select the study species as human (“Homosapiens”), set the confidence to be >0.4, hide disconnected nodes in the network, and use the downloaded.tsv file in String with R 4.0.4 language software is processed, and the PPI diagram is drawn according to the degree value (degree); the top 20 PPI core gene targets are finally obtained.The String database (https://david.ncifcrf.gov/), GO functional annotation and KEGG pathway enrichment analyses were performed on the intersection target genes of Zanthoxylum bungeanum and DM. The species was limited to “Human,” and the enriched entries with q values  less than 0.05 were retained and sorted according to the size of the q values. The R 4.0.4 language software was applied to the obtained genes for GO and KEGG enrichment analyses and visualization. The GO analysis selects the top 10 enrichment results for display, and the KEGG analysis selects the top 20 enrichment results for display, respectively.Based on the David database (Zanthoxylum bungeanum under item “1.1” was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and saved in “sdf” format as a small molecule ligand. We download the 3D structure of the protein corresponding to the core target from the RSCBPDB database (https://www.rcsb.org/) and save it in pdb format as a protein receptor. We use Discovery Studio 2019 software to process small molecule ligands, remove original ligands and water molecules, and add hydrogen atoms to the protein receptors to preprocess and find active pockets and then mix the core target protein receptors. Molecular docking with protein ligands [Zanthoxylum bungeanum, which was displayed in 3D and 2D structures, respectively.The 2D structure of the active ingredient compound of  ligands  was done2 constant temperature incubator (Thermo Company), microplate reader , electrophoresis apparatus , electric blast drying oven , gel imaging analysis system , and SW-CJ-1FD/2FD ultra clean workbench (Jiangsu Suzhou Purification Equipment Factory) were used for the experiment.Fetal bovine serum and high sugar DMEM were purchased from Gibco Company. Trypsin, CCK8 kit, BCA protein concentration determination kit, rabbit anti-AKT1, IL6, HSP90AA1, FOS, and JUN were all purchased from Cell Signaling Technology and Beyotime Biotechnology, respectively. COZanthoxylum bungeanum (5 kg) was purchased from Jiangjin District, Chongqing, China, in May 2021. It was identified by Vice Professor Guowei Wang, College of Pharmacy, Southwest University of China. The voucher specimen (no. 20210015) has been deposited in the Herbarium of Anshun University. After being dried, Zanthoxylum bungeanum was extracted three times with 95% ethanol. The extract was evaporated to obtain a residue (0.5 kg), suspended in water, and fractionated with dichloromethane to obtain a Zanthoxylum bungeanum dichloromethane extract .(1) Cell Culture. Human hepatoma cell line HepG2 was purchased from the cell bank of Chinese Academy of Sciences. The frozen cells were quickly thawed, added to the culture medium, and centrifuged for 3 min; then, the supernatant was discarded, the cells were transferred to the culture bottle, and the prepared complete culture medium  was added, and the cells were cultured in a 37°C, 5% CO2 incubator. When the cells in the culture bottle fuse to 80%~90%, they can be digested with trypsin and transferred to a new culture bottle.(2) Cell Grouping and Treatment. Cell modeling was as follows: 38.5 mg of palmitic acid was dissolved in 1.5 mL of anhydrous ethanol to obtain a palmitic acid master batch at a concentration of 100 mmol/L. 0.5 mL of the master batch was diluted with high-sugar DMEM containing 1% BSA to obtain 10 mL of 5 mmol/L palmitic acid solution. After filtration and debacterization, the cells were incubated for 24 h with DMEM containing 35 mmol/L glucose to obtain 0.25 mmol/L of the modeling agent.μmol/L, respectively.Grouping and drug administration was as follows: the control group (Ctrl) was given culture medium, the model group (Mod) and ZBE group were incubated in the incubator for 24 h after the intervention of the modeling agent by discarding the old medium and adding DMEM and ZBE extract with different concentration at 5, 10, 20, 40, and 80 (3) Cytotoxicity and Cell Viability Assay. The HepG2 cells were inoculated in 96-well plates at a density of 5 × 104 cells/well in a volume of 100 μL. After 24 h of cell culture, the cells were given a mold-making agent intervention for 24 h. The old solution was discarded, and the experimental groups were given different concentrations  of ZBE extract diluted in DMEM. After 24 h of administration, the medium was replaced with new medium; 10 μL of CCK8 assay solution was added to each well, incubated in the incubator for 30 min, shaken, and mixed; and the optical density value  was read at a wavelength of 450 nm on an enzyme marker to calculate the cell growth rate.(4) Western Blot Assay. The western blot method was used for AKT1, IL6, HSP90AA1, FOS, and JUN protein expressions in HepG2 cells. The HepG2 cells were inoculated in 6-well plates at a density of 2 × 105 cells/well. After successful modeling, ZBE was added at 20 μmol/L (low) and 40 μmol/L (high), respectively, for 24 h. RIPA lysis solution was added to each well for 220 μL, and the cells were lysed on ice for 10 min and scraped off and transferred to an EP tube, and the cells were repeatedly blown and broken in a cell crusher and continued to be lysed on ice for 10 min. The supernatant was extracted, and the protein concentration of each group was determined using the BCA kit, diluted and leveled using 5× protein loading buffer, and denatured in boiling water. The protein sample volume of 40 μg per well was added to the precast gel, and electrophoresis buffer was added for electrophoresis . The proteins were transferred to the PVDF membrane, blocked with 5% BSA for 1 h. The primary antibody (1 : 1000) was added overnight; the membrane was washed three times with TBST and incubated with goat anti-rabbit secondary antibody (1 : 1000) for 1 h. The membrane was washed three times with TBST and washed in ECL luminescent solution for color development and photographed, and the grayscale values of the bands were counted.(5) Statistical Analysis. Statistical analysis was carried out using SPSS 20.0 software. The statistical significance of the differences was measured between groups by ANOVA, and all the data were statistically analyzed by mean ± S.D. P < 0.05 was considered statistically significant, and P < 0.01 was considered to be significantly different.Zanthoxylum bungeanum met the screening criteria, including kokusaginin, skimmianin, diosmetin, beta-sitosterol, and quercetin, respectively , and the top 10 entries by Q-Value were visualized separately . Compared with the model group, the protein expression in the low-concentration group  and the high-concentration group (P < 0.01) of ZBE significantly decreased. The results showed that DM could be significantly improved by downregulating the expression of related proteins  after being treated with different concentrations (20 μmol/L and 40 μmol/L) of ZBE  known as AKT kinases, and it regulates many processes, including metabolism, proliferation, cell survival, growth, and angiogenesis. AKT is an important target kinase downstream of PI3K. PIP3 can bind to AKT, transfer AKT from the cytoplasm to the cell membrane, and phosphorylate Ser473 and Thr308 to fully activate AKT, thereby initiating the PI3K/AKT signaling pathway . It was he liver . Inflammhe liver . The Zanand IL-6 . It is sed death . It was ed death . C-JUN ped death . The inffunction .Zanthoxylum bungeanum mainly including kokusaginin, skimmianin, diosmetin, beta-sitosterol, and quercetin. The findings indicated that AKT1, IL6, HSP90AA1, FOS, and JUN are the most likely targets for Zanthoxylum bungeanum on treating DM. Experiment verification results revealed that DM could be significantly improved by downregulating the expression of AKT1, IL6, HSP90AA1, FOS, and JUN proteins after administrated 20 μmol/L and 40 μmol/L of ZBE. Zanthoxylum bungeanum is an effective drug in treatment of DM related to above core targets. The present study preliminarily clarifies that Zanthoxylum bungeanum may treat DM through multitargets and multipathways, which provides a new idea and points out a new direction for further experimental research and clinical application.Taken together, the active components of


Objective: Using a network pharmacological approach, this study will evaluate the effect of Xuefu Zhuyu Decoction in the treatment of atherosclerosis.Methods: The data were imported into the STRING database to construct a protein-protein interaction network, and the network topology was analysed with the Bisogenet plug-in by Cytoscape 3.7.2. Using the R language Bioconductor platform, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for potential targets of Xuefu Zhuyu Decoction in the treatment of atherosclerosis were performed, and import the results were imported into Cytoscape 3.7.2. To map the results and create a KEGG network diagram, we used Cytoscape 3.7.2 for analysis.Results: A total of 91 chemical components and 1320 disease targets were obtained, including 138 cross-targets. TNF, AKT1 and ALB were identified as important targets, and Gene Ontology functional analysis indicated that biological process was the primary cause of oxidative stress. The primary action of molecular function is binding. KEGG has explored and enriched 149 signalling pathways, including the AGE-RAGE signalling system and the TNF signalling network. According to a study involving molecular docking, quercetin and β-carotene have a strong binding affinity for AKT1 and ALB.Conclusion: The potential of Xuefu Zhuyu Decoction to treat atherosclerosis through multiple components and targets provides a way to further study its mechanism. Atherosclerosis (AS) is characterized by the accumulation of fat and fibrous particles, as well as calcification, in the principal arteries . The creUsing network pharmacology to investigate the mechanisms of TCM-disease interactions has proven useful. Through advanced target-disease interaction networks and bioinformatics research, network pharmacology, which is coherent with TCM as a whole, can identify potential mechanisms of prescription effects in disease therapy. This is because TCM components are intricate and multipurpose. Due to its virtual screening properties, however, it must be confirmed in cellular and/or animal experiments .Glycyrrhiza each, and Chai Hu). The active components of Xuefu Zhuyu Decoction were identified by screening in TCMSP, with an oral bioavailability (OB) of 30% and drug-likeness (DL) of 0.18. The likely protein targets were extracted from the Swiss target prediction database with conditional probability better than 0.1, and the screened protein targets were assigned standardized gene names in the UniProt database.Identification of active chemical constituents of Chinese herbal medicines using TCM systems pharmacology platforms TCMSP and TCMID , and their interactions were discovered. The indirect objectives were then acquired. The indirect targets have been added to the database of acting targets, which has been imported into the STRING database. To obtain the desired interaction network, Homo sapiens, who received a default score of 0.4, was utilized. Cytoscape 3.7.2 was used to analyze the network topology once the TSV format was imported and saved. After calculating the degree, betweenness centrality (BC), topological coefficient (TC), and closeness centrality (CC) of each node, the top 3 targets were selected as key target proteins.The possible targets of the antiatherosclerotic effect of Xuefu Zhuyu decoction were entered into the GeneMANIA database (https://www.r-project.org) and its backend database org, gene IDs (entrezIDs) of possible targets were determined. The Hs.e.g.db database was utilized to perform GO functional enrichment analysis on these possible targets, followed by the Bioconductor tools DOSE, cluster Profiler, and path-view. The GO function enrichment analysis of these potential targets included three components: DOSE, cluster Profiler, and the path-view instrument . We set p value Cutoff = 0.05 and q value Cutoff = 0.05 as appropriate for biological process (BP), cellular component (CC), and molecular function (MF). GO enrichment analysis revealed three fundamental categories: BP, MF and CC. The top ten items for enrichment are displayed as bar and bubble charts, with each category sorted in order of significance.Using R software (The targets of Xuefu Zhuyu Decoction acting on atherosclerosis were retrieved from the PDB database and saved in pdb format. After topological analysis, the compounds with the top 2 ligands in degree value were saved in mol2 format. The potential targets of Xuefu Zhuyu Decoction in atherosclerosis were molecularly docked with the main compounds of Xuefu Zhuyu Decoction using AutoDockTools-1.5.6.Xuefu Zhuyu Decoction includes 91 active constituents, as determined by screening criteria of OB 30% and DL 0.18, high GI absorption for pharmacokinetics, and over two “YESs” in druglikeness in TCMSP and TCMID. A total of 240 targets of Chinese medicine were retrieved by TCMSP and TCMID.Following the discovery of disease genes using GeneCards and Omim, 1320 atherosclerosis-related targets were identified, and targets with scores above the median were empirically classified as potential disease targets. Then, these targets were added to relevant targets found through searches of the OMIM database, and duplicate values were eliminated.Using the Venn tool in TBtools, Cytoscape 3.7.2 Active ingredients and an efficient target network of Xuefu Zhuyu Decoction. Software was used to calculate the topological parameters of the Xuefu Zhuyu Decoction network for the treatment of atherosclerosis to analyze the importance of activity components and action goals. Based on the findings, quercetin, β-carotene, and kaempferol may be the primary active components in Xuefu Zhuyu Decoction that help treat atherosclerosis by acting on multiple targets We uploaded the intersection targets of Xuefu Zhuyu Decoction and atherosclerosis to the STRING database using the Venn tool, with the confidence level set to 0.90, and then built the PPI network map of targets. Cytoscape 3.7.2 was utilized to create the protein network relationship map. To decide network placement, the degree value size is used. The degree value is proportional to the size of the node. Haematopoeia deemed AKT1, ALB, IL6, TNF, TP53, and other targets important targets for the treatment of atherosclerosis .GO annotation analysis of valid targets using R. The top ten results in BP, CC, and MF were selected, and oxidative stress was determined to be the major function of these target BPs. The primary functions of MF included binding activities. CC was predominantly active within the vesicle lumen, membrane raft, and microfilm areas. The top 20 KEGG-enriched signaling pathways visualized for analysis included the AGE-RAGE signaling route, the TNF signaling pathway, the IL-17 pathway, and others. The key components of Xuefu Zhuyu Decoction identified by topological analysis were molecularly docked with the probable targets of Xuefu Zhuyu Decoction in atherosclerosis using AutoDockTools-1.5.6. The likelihood of action increased as the ligand‒receptor binding configuration became more stable, and the degree value was used to identify the most crucially important targets. A binding energy of -4.25 kcal/mol or less indicates some binding activities between ligand small molecules and receptor proteins, 5.0 kcal/mol or greater indicates good binding activity between the two, and -7.0 kcal/mol or greater indicates robust conjugation activity between ligand and receptor. According to the docking results in Recent clinical research indicates that Chinese herbal medicine has special benefits for the development of anti-AS drugs and for enhancing the quality of patient survival. Several studies have already demonstrated the anti-inflammatory and antioxidant properties of herbal therapy in the aetiology of AS-7. Despite the fact that long-term use of statins can cause undesired side effects such as drug dependence and liver damage, oral statins are commonly used in the prevention and treatment of atherosclerosis . Xuefu ZThis study screened 91 active compounds using a combination of bioinformatics and network pharmacology, providing 240 active ingredient action targets. In addition, 1320 disease gene targets were intersected to produce a total of 138 common targets, among which active components such as quercetin, -carotene, and kaempferol were revealed. This study provides a molecular mechanism for the use of Xuefu Zhuyu Decoction in the treatment of atherosclerosis. The findings of this study provide a more detailed explanation of how Xuefu Zhuyu Decoction’s can treat atherosclerosis at the molecular level and can be applied to make medicines to flight atherosclerotic-related illnesses.A topological analysis of the ingredient-target-pathway network revealed that quercetin and β-carotene are key active ingredients in the treatment of atherosclerosis and correlate with more targets. It has been reported that quercetin, which blocked the Galectin-3-NLRP3 signaling pathway, reduced the inflammatory response caused by atherosclerosis . MoreoveAKT1 can participate in numerous signaling pathways, such as the cell cycle, inflammatory response, PI3K/Akt, MAPK, and AGE-RAGE signaling . NumerouAccording to the results of the GO analysis, the therapy of the atherosclerotic pathway by Xuefu Zhuyu Decoction predominantly includes lipid metabolism and binding. Xuefu Zhuyu Decoction may aid in the treatment of AS by regulating the AGE-RAGE signaling cascade, TNF, and MAPK, according to a KEGG analysis.via quercetin, β-carotene, kaempferol, and other flavonoid active component monomers. Consequently, it provides evidence to support its pharmacological impact. However, Network pharmacology is based on databases and analytical software, this study was analyzed through two databases, so the analysis results have limitations. The additional experimental validation is necessary to substantiate this assertion. In addition, the concentration used is not recorded in the database, but for the drug to work, it needs to be used at a certain concentration.A network pharmacology approach was utilized to evaluate the therapeutic targets and mechanisms of action of core Chinese herbal medicines on common-type atherosclerosis. Xuefu Zhuyu Decoction has anti-inflammatory and antioxidant properties