Kaixinsan (KXS), a traditional Chinese medicine formula, has been demonstrated to be effective in the treatment of depression. The present study applied a network pharmacology approach to dig out the new targets and mechanism of action of KXS and the active compounds in the treatment of depression.  A network pharmacology approach based on public databases including ADME  evaluation, targets prediction, construction of networks, and molecule docking was used and validated the predicted new antioxidant targets and mechanisms in vitro. Based on an in vitro experiment, we verified the AKT1/Nrf2 pathway related to the thioredoxin (Trx) antioxidant mechanism. 2O2 induced SH-SY5Y cell damage model, this showed kaempferol and ginsenoside rh2 could enhance the activity of the Trx system by upregulation of AKT1 to activate Nrf2 in vitro.  The present study sorted 31 pharmacologically active components  through the ADME algorithm from KXS. 136 potential molecular targets  were predicted, of which there were 69 targets clearly related to depression. By compound-depression targets (C-DTs) network constructed, and protein-protein interaction networks (PPI) and KEGG pathway enrichment analyzed, we identified active compounds mediating depression-related targets to exert synergism on the predictive AKT1/Nrf2 pathway related to thioredoxin (Trx) antioxidant mechanism and other inflammation-related signaling pathways as well as neurotransmitter related signaling pathways. In the H Taken together, by comprehensive systems pharmacology approach analysis, we found that KXS and its active compounds might exhibit antioxidant effects by stimulating the AKT1/Nrf2 pathway in the treatment of depression, which might shed new light on innovative therapeutic tactics for the new aspects for depression in traditional Chinese medicine in future studies. Depression has huge social risks of high incidence, disability, and suicide. At present, the main clinical antidepressants are selective 5-HT reuptake inhibitors, tricyclic or tetracyclic antidepressants, and monoamine oxidase inhibitors. Chronic treatment with antidepressants is only 60 to 70 percent effective with significant side effects .With a long history of thousands of years, traditional Chinese medicine (TCM) plays important roles in the treatment of mental illness. Natural products, such as plants, animals, and minerals, are typically prescribed in TCM, in which herbal or botanical products are most widely used. Depression can be traced back to the traditional Chinese medicine book “Huang Di Nei Jing” more than 2000 years ago, which belongs to the category of “depressive syndrome-YuZheng” in TCM. TCM shows its advantage for containing a variety of chemical components, which targets through various channels, and significantly alleviating complications such as insomnia and anxiety while treating depression.In fact, the serotonergic, dopaminergic, and noradrenergic systems are deeply involved in the mechanisms of TCMs' antidepression process. Among them, inhibition against monoamine oxidase reaction and increasing synaptic availability of monoamines are the main factors in TCMs' monoamine regulation mechanism. For example, Xiaoyaosan could improve the serotonergic metabolism by regulating the expression levels of tryptophan hydrogenase 2 (TPH2) and indoleamine 2,3-dioxygenase 1 (IDO1), thus exerting an antidepressant effect . Other tGinseng Radix (Panax ginseng C. A. Mey.), Polygalae Radix , Poria (Poria cocos (Schw.) Wolf), and Acori Tatarinowii Rhizoma (Acorus tatarinowii Schott), with different ratio. As our previous clinical trials showed that KXS (3 : 3 : 2 : 2 ratios) ameliorated clinical symptoms of patients with minor and modest depression [Ginseng Radix [Polygalae Radix [Acori Tatarinowii Rhizoma [Poria [KXS is a classic ancient prescription, first recorded the “Qianjin Yaofang” written by Sun Simiao in the Tang Dynasty. It is composed of pression . Pharmacpression . Althougng Radix , 10, Polae Radix , Acori T Rhizoma  and Poria [Poria  have an Herein, we screened the potential active compounds of the four herbs contained in KXS with the computational ADME and selected depression-related targets, then combined the KEGG enrichment analysis and PPI were performed illustrate its possible mechanisms, especially those involved with oxidant stress, and finally performed to reveal the potential compounds in KXS on its specific antioxidant pathway. The work scheme of this research is shown in https://tcmspw.com/tcmsp.php) [http://119.3.41.228:8000/tcmid/search/) [https://www.symmap.org/) [Ginseng Radix, Polygalae Radix, Poria, and Acori Tatarinowii Rhizoma contained in KXS were screened. According to the ADME algorithm, oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18 were set as the screening criteria, and the active compounds, MolID, and action targets of the herbs were finally obtained. The same repeated active compounds and their action targets in the four herbs were combined using Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/) online software to obtain the effective compounds and targets in the KXS.According to the database TCMSP (msp.php) , TCMID (search/) , and Symap.org/) , the effhttps://www.disgenet.org) [https://www.uniprot.org/) [https://string-db.org/), the obtained common targets of KXS in the treatment of depression were visualized, and the protein-protein interaction networks (PPI) were drawn.The Disgenet (net.org)  databaseot.org/)  databasehttps://www.kegg.jp/) [P < 0.05 was set, the species was limited to homo sapiens, the top 10 targets related to KEGG pathway enrichment were listed, and the antioxidant pathways were found.The KEGG database (egg.jp/)  conducteThe Autodock Vina software calculated the free energy of molecular docking between the selected key targets and the corresponding compounds of KXS. The Pymol software was used for mapping, and the results of molecular docking were visualized to verify the network pharmacological analysis.5/mL and a volume of 100 μL in the logarithmic growth phase was added to the 96-well plate. After screening the H2O2 induced damage concentration, it is set on 100 μmol/L to induced injury model. Then, the active compounds kaempferol and ginsenoside rh2 with different dosages were added to the cell for 24 h. Four groups were set up: untreated control group, H2O2 induced injury model group, kaempferol treatment group, and ginsenoside rh2 treatment group.The human neuroblastoma SH-SY5Y cells were selected to construct the model of oxidant stress damage in vitro. The cell-complete medium was DMEM supplemented with 10% fetal bovine serum. SH-SY5Y cell suspension with a concentration of 5 × 105) were cultured in an incubator with 5% CO2 at 37°C for 24 h under different treatments in the 96-well plate. Totally 10 μL CCK8 solution was added into each well, and the cells were cultured for another 1 h. The absorbance was finally determined at 450 nm with a microplate reader.The cell viability was measured with the Cell Counting Kit-8 (CCK8) Assay (Beyotime). The SH-SY5Y cells , Nrf2 , Trx  were incubated overnight, and then incubated the (HRP)-conjugated secondary antibody . Immunoblots were visualized by BeyoECL Plus kit  and quantified by densitometry in the western blotting detection system  and ImageJ software .The SH-SY5Y cells were inoculated in the six-well plate after four ways of treatment, and 100 The activity of TrxR was measured by the Solarbio thioredoxin reductase activity Kit. We used 2-vinylpyridine to inhibit the original reduced glutathione in each sample. The activity of TrxR was calculated by measuring the absorbance at 412 nm.P < 0.05 represented statistical significance.Statistical data were analyzed by GraphPad Prism 5 and compared with a one-way analysis of variance. The bilateral 95% confidence interval was used for all tests, and a value of Ginseng Radix were screened, and 117 targets were identified. 6 effective compounds of Poria were screened, and 25 targets were identified. 4 effective compounds of Acori Tatarinowii Rhizoma were screened, and 81 targets were identified. 5 effective compounds of Polygalae Radix were screened, and 31 targets were identified  signaling pathways were preserved for further analysis. Multiple signaling pathways were involved in the treatment of depression with KXS, including pathways in cancer and metabolic pathways, and neurotransmitter-related pathways  in 24 h induced a significant increase of TrxR activity, compared with the control group, and then decreased at 72 h, which might be a process of depletion. Kaempferol and ginsenoside rh2 kept the activity of TrxR from 24 h to 72 h, compared with H2O2 induced damage group, indicating they might keep the Trx protein system maintained for stronger antioxidant capacity for a longer time. And the antioxidant effect of kaempferol was significantly better than that of ginsenoside rh2.In According to the retrieval of Trx protein-related pathways in the KEGG database, AKT1 and Nrf2 were the upstream proteins in the antioxidant pathway of Trx. Here, the results indicated that kaempferol and ginsenoside rh2 could increase the expression of Trx protein. Meanwhile, these two monomer compounds could also increase the expression of AKT1 and Nrf2, which boosted the targets in the AKT1/Nrf2/Trx pathways cascade working together. Similar to Trx activity, kaempferol works better than that of ginsenoside rh2 in the AKT1 /Nrf2/Trx pathways .According to our results, by systems pharmacology approach which contained ADME screening, targets prediction, network analysis, and pathway screening, we verified the “multicompound, multitarget, and multipathway” properties of the KXS formula. We identified depression-associated targets of KXS compounds, among them AKT1 was identified as core antidepressive target of KXS. More specifically with its antioxidant mechanism, we found that KXS and its anti-oxidant monomer compounds might work on AKT1/Nrf2 pathways to activate the Trx antioxidant system. According to the results, the advantages of KXS for treating depression mainly concentrate on the following aspects.α and IL-6 in the serum of depressed mice, and up-regulate the activity of superoxide dismutase (SOD) in the hippocampus [Firstly, the active compounds of the KXS formula are large in number, which might greatly increase the multitarget effect and reduce the possibility of side effects and drug resistance. KXS takes flavonoids, saponins, oligosaccharides, polysaccharides, and volatile oil as the main active compounds , and itspocampus . These rβ, IL-6, and TNF-α [β-protein (Aβ) [Secondly, many studies had shown that the mechanism of KXS in the treatment of depression was to improve the activity of neurotransmitters such as 5-HT  and acetnd TNF-α , and incein (Aβ) , improveein (Aβ) , inhibitein (Aβ) . These sein (Aβ) , reactivein (Aβ) , heme oxein (Aβ) , and Trx2O2 oxidative damage of SH-SY5Y cells in vitro, and instantly keeping enhanced the activity of Trx by increasing its upstream proteins AKT1 and Nrf2. While, the antioxidant effect of kaempferol is better than that of ginsenoside rh2, which makes kaempferol become a valuable active compound with new targets for treating depression. Of course, there are some deficiencies in this study. For example, the components and effective targets of KXS after screening by ADME are relatively few, and only in vitro model studies have been carried out. There is still a lot of work to be made up in the following studies.Thirdly, most studies on the antioxidation of antidepressants focused on the GSH system, ROS, and HO systems. The mechanism of antioxidants was mainly on Trx antioxidant system, relatively less studied. The Trx system is a small molecule protein system associated with redox action in the body. This system is composed of Trx, thioredoxin reductase (TrxR), and nicotinamide adenine dinucleotide phosphate (NADPH). It can reduce the cysteine residues of oxidized Trx and further acts as an electron donor for ribonucleotide reduction, maintaining its antioxidant capacity , so thatIn conclusion, for the first time, we identified the AKT/Nrf2 pathway and targets of KXS compounds using an integrative systems pharmacology approach. Furthermore, we validated the mechanisms by which KXS and its active compound ameliorated depression as shown in


A growing number of disorders has been associated with mutations in the components of the vesicular transport machinery. The early secretory pathway consists of Endoplasmic Reticulum, numerous vesicles, and the Golgi Complex (GC), which work together to modify and package proteins to deliver them to their destination. The GC is a hub organelle, crucial for organization of the other secretory pathway components. As a consequence, GC’s form and function are key players in the pathogenesis of several disorders. Skeletal muscle (SKM) damage can be caused by defective protein modifications and traffic, as observed in some Limb girdle muscular dystrophies. Interestingly, in turn, muscle damage in Duchenne dystrophic SKM cells also includes the alteration of GC morphology. Based on the correlation between GC’s form and function described in non-muscle diseases, we suggest a key role for this hub organelle also in the onset and progression of some SKM disorders. An altered GC could affect the secretory pathway via primary , or secondary mechanisms , which converge in SKM cell failure. This evidence induces considering the secretory pathway as a potential therapeutic target in the treatment of muscular dystrophies. In recent years, study of the correlation between the secretory pathway and inherited human diseases has become increasingly important, allowing for a link to a growing number of disorders and mutations in the components of the secretory pathway ,2,3.In the eucaryotic cell, the secretory pathway is constituted by several components with the fundamental tasks to receive, modify, and package proteins and lipids to drive them to their final destinations. In the conventional secretion pathway, proteins are synthetized in the rough endoplasmic reticulum (ER), they leave the ER at the level of the ER- exit sites (ERES) and are transported in COPII vesicles to the Golgi Complex (GC), where they undergo post-translational modifications  and are packed into post-GC vesicles to reach their target. Defective proteins return to the ER via COPI vesicles. The early secretory pathway, which consists of the ER, ERES, COPII and COPI vesicles, and the GC is the first secretory compartment that the proteins transit to reach their final destination. Since this part of the secretory pathway fulfill the task of selectively transporting native proteins to reach their final target, the ER to GC interface is a fundamental checkpoint for protein scrutinization .In mature skeletal muscle (SKM) cells, the architectural disposition of the early secretory components is unique: the GC is formed by very small elements, localized near the nucleus and in the fiber in close association with ERES in a fiber-type dependent fashion ,6,7. ThiActually, these data agree with the evidence that, in other cell types, the GC is not only the central organelle for glycosylation, but it exerts a hub function in the regulation of the transport via the modulation of the assembly and localization of vesicles, as well as ERES distribution . This prCongenital muscular dystrophies are a heterogeneous group of inherited disorders characterized by progressive muscle weakness and wasting ,13. AlthInterestingly, despite the numerous proteins involved in the early secretory pathway, the very low level of mutations associated with a disease indicates its essential role in the cell physiology. Accordingly, the literature reports several forms of muscular dystrophies and myopathies associated with mutations to genes involved in post-translational modifications  iIn general, due to the ubiquitous expression of most of these proteins, the damage to the SKM tissue can be one of the features in a very complex clinical condition, as largely described for some alpha-dystroglycanopathies ,14,15 ancis region facing the ER and trans region directed toward the plasma membrane [cis face of the GC (anterograde traffic), where they are modified and released via the trans face, to be then directed to the plasma membrane. Defective proteins are transported in COPI vesicles in the retrograde traffic pathway [The GC is involved in protein quality control and is the fundamental organelle for the glycosylation of numerous proteins. To this aim, the GC is a highly dynamic organelle, organized in flattened polarized cisternae, organized in a membrane ,24,25. T pathway .There is convincing evidence that the maintenance of the GC structure is important for its own function and that several diseases are correlated with modifications of the GC morphology and localization ,11,25. IA comparable role for the GC could be also advanced in the context of the SKM cell because, as suggested by Kanagawa in a description of alpha-dystroglycanopathies , proteinIn support to the important role of the GC in the SKM cell, our recent work showed that in in vitro differentiating myoblasts, the depletion of GM130 interferes with GC morphology and ERES organization. It strongly inhibits the transport of M-cadherin, a membrane protein involved in myoblast fusion , to the Interestingly, it has been reported that mutations to the GC matrix protein GM130, in a pediatric patient from consanguineous parents, induce developmental delay, neuromuscular disorders and muscular damage . Work frAlthough GM130 is a ubiquitous protein, and therefore the clinical picture is complicated with the malfunctioning of multiple organs and system  ,29, in vOn the other hand, it has been shown that the alteration of muscle specific proteins, not directly involved in the early secretory pathway, could also have an impact on GC form and function. This is the case of the Duchenne muscular dystrophy (DMD), where the loss of dystrophin is accompanied by the alteration of the morphology and mis-localization of the GC, ,31, withIn conclusion, in light of the above-mentioned correlation between GC form and function in non-muscle cells ,11,27,33Either for direct or secondary contributions, the resulting effect of a non-working early secretory pathway is an accumulation of proteins . Similar to an airways traffic jam, where the passengers take alternative flights to reach their final destination, we can try to manipulate some of the components of the intracellular traffic by exploiting alternative routes and/or hasten the traffic, allowing proteins to reach their destination. Potentially, different approaches can be applied to reduce the consequences of a defective secretory pathway . These sSome compounds have already been identified ; howeveIt can be suggested that the application of secretory pathway regulators could be of help in also treating muscular dystrophies. Evidently, the choice of the treatment depends on the type of disease and whether the secretory pathway defects result from a direct  or secondary effects . Actually, in the latter situation, the therapeutic targeting of the secretory pathway is not sufficient to prevent or delete the principal effects of a defective protein , but could have an adjuvant effect by modulating the load of the above-mentioned secondary effects .However, since the different components of the secretory pathway are highly connected, it is not excluded that a single strategy can have multiple effects and can be helpful for several muscular pathologies.In this perspective, we briefly reported and discussed data on the relevance of the early secretory pathway and its hub organelle, the GC, in the context of muscular dystrophies. Despite the paucity of data on the secretory pathway in the SKM cell, observations from the literature lead us to suggest that the integrity of the GC and vesicular components is necessary for maintenance of the form and function of this tissue. Based on observations in other cell types, where the alteration of the secretory pathway function results in unproper targeting and the accumulation of proteins, we hypothesize that the secretory pathway can be a potential therapeutic target in the treatment of SKM diseases. A desirable outcome of future research is to gain further understanding of mechanisms regulating the secretory machinery in the SKM and to test the possibility of targeting the secretory pathway to ameliorate SKM function in pathologic conditions.


Polygonum cuspidatum Sieb and has been demonstrated to exert anti-inflammatory and neuroprotective activities. In the present study, we aimed to explore the therapeutic mechanisms of PD against chemotherapy-induced neuropathic pain.  Polydatin (PD) is the primary active compound in  The putative targets of PD were obtained from the CTD and SwissTargetPrediction databases. Neuropathic pain- and VIN-related targets were collected from the CTD and GeneCards databases. Subsequently, the intersection targets were obtained using the Venn tool, and the protein-protein interaction (PPI) was constructed by the STRING database. GO and KEGG enrichment analyses were performed to investigate the biological functions of the intersection targets. Further, a rat model of VIN-induced neuropathic pain was established to confirm the reliability of the network pharmacology findings.  A total of 46 intersection targets were identified as potential therapeutic targets, mainly related to neuroinflammation. KEGG pathway analysis indicated that the IL-17 signaling pathway was involved in the mechanism of the antinociceptive effect of PD. PPI network analysis indicated that RELA, IL-6, TP53, MAPK3, and MAPK1 were located at crucial nodes in the network. Additionally, PD exerted an antinociceptive effect by increasing the nociceptive threshold. The results of qRT-PCR, western blot, and immunohisochemistry indicated that PD inhibited the IL-6, TP53, and MAPK1 levels in VIN-induced neuropathic pain rats.  Overall, this research provided evidence that suppressing inflammatory signaling pathways might be a potential mechanism action of PD's antinociceptive effect against VIN-induced neuropathic pain. Neuropathic pain is a chronic secondary pain, which is characterized by shooting pain or spontaneous persistent and induced magnified pain responses following harmful or non-harmful stimuli . VincrisPolygonum cuspidatum Sieb, a traditional Chinese herbal in China. Previous reports have demonstrated multiple pharmacological effects of PD such as anti-inflammatory, neuroprotective, antioxidative, immune-regulating, cardioprotective, and antiplatelet aggregation activities [Polydatin (PD), also known as polygonin, is a stilbenoid originally extracted from the root of tivities , 11. PD tivities . PD exhitivities . PD alsotivities . Moreovetivities . PD exertivities . All theBased on these studies, we performed network pharmacology analysis to screen the hub genes and potential therapeutic mechanisms of PD against VIN-induced neuropathic pain. Besides, the analgesic effect of PD associated with the suppression of inflammatory genes level was verified by animal experiments.https://pubchem.ncbi.nlm.nih.gov/). The comparative toxicogenomics (CTD) database is a premier public resource that advances understanding about human health and chemical exposures [https://ctdbase.org/) and SwissTargetPrediction databases (http://www.swisstargetprediction.ch/) to obtain the PD-related targets.The 2D chemical structure of PD was downloaded from the PubChem Database (xposures . SwissTaxposures . We usedhttps://www.genecards.org/). Then, the intersection targets of PD, VIN, and neuropathic pain were visualized by using the Venn online tool (https://bioinfogp.cnb.csic.es/tools/venny/index.html).The VIN-related and neuropathic pain-related targets were collected from the CTD database and GeneCards database (https://cn.string-db.org/), with species limited to “Homo sapiens” and the highest confidence (0.9). Then, the TSV format file was downloaded and imported into Cytoscape software (3.8.0) to visualize the PPI network. The key genes were screened according to stress, betweenness, closeness, degree, DMNC, EPC, MNC, and radiality, and the intersection genes were identified as the hub genes. Besides, a compound-targets-pathways network was constructed via the Cytoscape software (3.8.0) [The intersection genes were uploaded to the String 11.5 ( (3.8.0) .https://metascape.org/gp/index.html) to obtain KEGG data and GO biological process data. Then, the clusterProfiler package of R software was used to visualize the results of the KEGG pathway analysis and GO enrichment analysis.Metascape could provide a comprehensive gene list annotation and analysis resource for experimental biologists . The intad libitum. The animal experimental protocol was approved by the Animal Experimental Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University, and the experimental procedure was performed by the WHO guidelines for animal care.Male Sprague-Dawley rats (190-230 g) were obtained from the Animal Laboratory Center of Xinjiang province and housed under a specific pathogen-free environment  with food and water Animals were acclimated for one week before induction of chemotherapy pain. VIN was dissolved in sterile saline (0.9% NaCl). The rats from the model group were injected intraperitoneally with VIN (0.1 mg/kg) for ten days (in a two-five days cycle with two days pause) , while rMechanical allodynia was performed on days 0, 7, 14, and 21 according to a previous report . Rats weThermal hyperalgesia was performed on days 0, 7, 14, and 21 according to a previous report . Rats weα, IL-6, IL-1β, and IL-17) and macrophage marker (CD163) in the supernatant were measured by ELISA kits  based on the manufacturer's protocols. The catalog numbers of inflammatory cytokines and macrophage marker were as follows: IL-6 (BMS231-2), TNF-α (BMS2034), IL-17 (BMS6001TEN), CD163 (88-50361-22), and IL-1β (BMS224-2).After the last nociceptive behavioral tests, the animals were anesthetized with 50 mg/kg of pentobarbital sodium. L4-L6 dorsal root ganglion sections were harvested and homogenized on ice using a homogenizer. Afterward, the homogenate was centrifuged , and the supernatant was collected. The levels of inflammatory factors  based on the manufacturer's protocols. Two microgram of RNA was used to synthesize cDNA by cDNA synthesis kits . qRT-PCR was carried out by using a CFX384 Real-Time System C100 Thermal Cycler (Bio-Rad) based on the manufacturer's protocols. Primer sequences used in the present experiment were presented in μg of protein was separated by 10% SDS-PAGE and transferred onto the PVDF membrane (Roche). Then, the blot was blocked with defatting milk powder (5%) at room temperature for one hour. After that, the blot was incubated overnight with the following primary antibodies: anti-IL-6 , anti-MAPK1 , anti-TP53 , and anti-GAPDH . After incubation, the blots were incubated with horseradish peroxidase-labeled secondary antibody for 60 min at room temperature. Following incubation, the blots were measured by a chemiluminescence reagent , and the band intensity quantification was performed using ImageJ software (NIH). GAPDH was used as an endogenous control.We carried out the western blot analysis based on the standard procedure in previous reports. The dorsal root ganglion tissues were homogenized in cold RIPA lysis buffer with protease inhibitors. After centrifugation at 10,000 g for 20 min, the supernatant was collected for protein quantification. 10 μm paraffin sections of dorsal root ganglion was deparaffinized using xylene and rehydrated using a gradient of ethanol. Endogenous peroxidase was suppressed using H2O2 (3%) for 0.5 h. Then, the slices were incubated with normal goat serum (10%) and anti-IL-6  or anti-TP53  or anti-MAPK1  primary antibodies at 4°C overnight. The slices were washed twice in PBS and incubated with a goat anti-rabbit antibody (1 : 200) at room temperature for 60 min. Subsequently, slices were visualized using a DAB reagent. Finally, we used light microscopy to obtain immunohistochemistry images.The dorsal root ganglion tissues were soaked in 4% formalin overnight. 5 P < 0.05.Data were statistically analyzed using GraphPad Prism 5 software and presented as mean ± SD. The results from behavioral tests were analyzed with repeated measures analysis of variance (ANOVA). Comparisons of results between groups were performed using one-way ANOVA followed by Bonferroni's test. Significance was set at The PD's 2D structure was shown in The top 10 GO terms of biological process (BP), cellular component (CC), and molecular function (MF) were presented in The top 15 most significantly enriched KEGG pathways were presented in The top 10 key genes were selected based on the 8 classification methods  in cytoHubba  to screeWe constructed a compound-targets-pathways network using Cytoscape software (3.8.0) to obtain a visual analysis result. As shown in P < 0.05). A high dose of PD treatment effectively inhibited VIN-induced neuropathic pain via increasing paw withdrawal threshold (28.03 ± 1.87 g) and paw withdrawal latency (10.8 ± 1.43 s) (P < 0.05).As shown in α (235.6 ± 23.08 pg/mg prot), IL-6 (166.9 ± 15.36 pg/mg prot), IL-1β (175.3 ± 15.41 pg/mg prot), IL-17 (101.1 ± 9.32 pg/mg prot), and CD163 (125.3 ± 11.25 pg/mg prot) in the VIN group compared to those in the control group (P < 0.05). A high dose of PD treatment significantly inhibited TNF-α (155.9 ± 27.87 pg/mg prot), IL-6 (111.4 ± 11.75 pg/mg prot), IL-1β (125.2 ± 21.13 pg/mg prot), IL-17 (74.98 ± 12.51 pg/mg prot), and CD163 (92.10 ± 7.92 pg/mg prot) in VIN+HPD compared to those in the VIN group (P < 0.05).As shown in P < 0.05). A high dose of PD treatment downregulated the expression of IL-6 (2.03 ± 0.21), TP53 (2.08 ± 0.54), and MAPK1 (1.66 ± 0.34) in the VIN+HPD group (P < 0.05). Besides, VIN injection upregulated the protein expression of IL-6 (3.23 ± 0.51), TP53 (3.16 ± 0.70), and MAPK1 (4.16 ± 0.45) in the VIN group compared to those in the control group (P < 0.05) (P < 0.05), which was consistent with the above findings. We also performed the immunohistochemical experiment to further validate the above results. As shown in P < 0.05). However, the expression levels of IL-6 (31.30 ± 7.59%), TP53 (32.97 ± 5.35%), and MAPK1 (31.73 ± 2.95%) in the VIN+HPD group were lower than those of the VIN group (P < 0.05).We verified the expression of the three hub genes using qRT-PCR analysis and western blot analysis to further clarify the potential mechanism of PD against VIN-induced neurotoxicity. As shown in  < 0.05) . A high Chemotherapy-induced neuropathic pain is a complex chronic disease, which is caused by damage to the nervous system . ChemothIn the present study, the network pharmacology and animal experimental approach were performed to reveal the potential therapeutic mechanisms of PD in the treatment of VIN-induced neurotoxicity. First, we identified 46 potential targets using the public databases. The results of KEGG enrichment analysis showed that these target genes were mainly involved in the IL-17 signaling pathway and cytokine-cytokine receptor interaction, which were closely associated with the pathologic process of chemotherapy-induced neuropathic pain. Based on the results of GO-BP enrichment analysis, these target genes mainly focused on the cellular response to lipopolysaccharide, cytokine-mediated signaling pathway, positive regulation of leukocyte cell-cell adhesion, humoral immune response, neuroinflammatory response, etc. In addition, we also identified five hub genes  as the most promising candidate targets of PD acting on the progression of chemotherapy-induced neuropathic pain.β, and TNF-α) [in vivo experiment further indicated that PD decreased the gene expression of IL-6 in VIN-induced neuropathic pain, implying that IL-6 was the potential therapeutic target of PD against VIN-induced neuropathic pain.Neuroinflammation is a potential codriver of chemotherapy-induced neuropathic pain. It has been reported that the chemotherapy-induced increase in inflammatory factors and a close relationship with the occurrence and development of neuropathic pain , 30. Macd TNF-α) . These md TNF-α) . In the d TNF-α) . Besidesd TNF-α) . IL-17 id TNF-α) . Moreoved TNF-α) . To suppd TNF-α) . In our in vivo experiment further indicated that PD regulated the gene expressions of TP53 and MAPK1 in VIN-induced neuropathic pain, suggesting that TP53 and MAPK1 were the potential therapeutic targets of PD against VIN-induced neurotoxicity. One of the limitations of this study is the species difference between rodent pain models and clinical chemotherapy-induced neuropathic pain. To overcome this hurdle, the use of species that are closer to humans than rodents, such as nonhuman primates, could hence understand the neuropathic pain and improve the passage of new therapies through clinical testing.TP53, a major neuronal proapoptotic gene, was implicated in synaptic terminal injury and apoptosis, and its activation was associated with the etiopathogenesis of Parkinson's disease , 39. It In conclusion, our study firstly demonstrated the therapeutic effects of PD on VIN-induced neurotoxicity via network pharmacology and experimental verification. These findings indicated that PD alleviated VIN-induced neurotoxicity via downregulation of IL-6, TP53, and MAPK1 expressions. This study provided a novel approach to exploring the potential therapeutic mechanism of PD in the treatment of neuropathic pain.


Panax Ginseng and Ginkgo biloba extracts (CGGE) is an effective treatment for nervous system diseases, but the neuroprotective mechanism underlying CGGE remains unclear. Both network analysis and experimental research were employed to explore the potential mechanism of CGGE in treating ischemic stroke (IS). Network analysis identified a total number of 133 potential targets for 34 active ingredients and 239 IS-related targets. What’s more, several processes that might involve the regulation of CGGE against IS were identified, including long-term potentiation, cAMP signaling pathway, neurotrophin signaling pathway, and Nod-like receptor signaling pathway. Our studies in animal models suggested that CGGE could reduce inflammatory response by inhibiting the activity of Nod-like receptor, pyrin containing 3 (NLRP3) inflammasome, and maintain the balance of glutamate (Glu)/gamma-aminobutyric acid (GABA) via activating calmodulin-dependent protein kinase type Ⅳ (CAMK4)/cyclic AMP-responsive element-binding protein (CREB) pathway. These findings indicated the neuroprotective effects of CGGE, possibly improving neuroinflammation and excitotoxicity by regulating the NLRP3 inflammasome and CAMK4/CREB pathway.Stroke is a major cause of death and disability throughout the world. A combination of  Stroke is one of the leading causes of mortality and disability, especially in low-income and middle-income countries . Accordi2+ influx. The increased intracellular Ca2+ causes mitochondrial dysfunction and overproduction of reactive oxygen species (ROS), which activates inflammatory responses and results in the death of damaged neurons  and CREB (The underlying mechanism of ischemic stroke (IS) is complex. Excitotoxicity and inflammation play crucial roles in the pathophysiological process of IS. Overactivation of the glutamate receptor mediates excitotoxicity, which has been suggested as a critical event in ischemic brain injury. Glutamate (Glu) is a major excitatory neurotransmitter in the central nervous system and acts on metabotropic and ionotropic receptors  to mediate excitatory synapse transmission . While g neurons . NMDAR a neurons . Tat-NR2and CREB . Nod-likand CREB . There iand CREB . The NLRand CREB .Panax Ginseng (P.ginseng C.A.Mey.) is a kind of valuable traditional Chinese medicine (TCM) with a history of thousands of years. The neuroprotective effects of panax ginseng on neurodegeneration diseases have been widely reported in the past decades, including cerebral ischemia, Alzheimer’s disease, and Parkinson’s disease  is a natural medicine widely used to treat neurodegenerative diseases. The neuroprotective effects of ginkgo biloba on ischemic brain injury are mainly through the regulation of excitotoxicity, inflammatory pathways and oxidative damage  showed beneficially neuroprotective effects against neuronal apoptosis , which is an effective treatment for vascular dementia  and G. biloba (NO.090914) were provided by the Shineway Pharmaceutical Group Co. Ltd. . Panax ginseng extract was prepared by adding eight times water to the powder of panax ginseng roots and rhizomes, extracting with boiling water for 3 h, then drying with a vacuum rotary evaporator at 80°C (50 g extract from 1 kg P. ginseng). Ultraviolet (UV) spectroscopy or high-performance liquid chromatography (HPLC)-UV method was used to determine the extract’s total active components and main ingredients to control the quality. The quantitative analysis showed that the total ginsenoside content was 77% in ginseng extract, among which ginsenoside Rb1, Rg1, and Re are13.1% (6.55 mg/g), 5.5% (2.75 mg/g), and 3.2% (1.6 mg/g), respectively . In the extract of ginkgo biloba, total flavonoids accounted for 49%, quercetin, kaempferol, and isorhamnetin occupied 28.7% (14.35 mg/g), ginkgolide A, B, C and bilobalide accounted for 11.6% (5.8 mg/g), and ginkgolide A was 3.3% (1.65 mg/g) (Extracts of ectively . Ginkgo 65 mg/g) . Both inAdult male SD (Sprague-Dawley) rats (200–220 g) were provided by the Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No. SCXK (Beijing) 2016-0011). All rats were housed at 25 ± 1°C temperature and 65% ± 5% humidity, with a 12 h light/dark cycle (lights on from 7:00 to 19:00). All rats had free access to food and water. The Ethics Review Committee for Animal Experimentation of Xiyuan Hospital, China Academy of Chinese Medical Sciences, approved the experimental protocols described in this study.n = 10): Sham group, MCE group, Tat-NR2B9C group (7.5 mg/kg), CGGE low group (15 mg/kg), and CGGE high group (30 mg/kg). The MCE rat model was established according to a previously described method  was used to identify and screen the active compounds of CGGE with a restriction of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 (https://www.uniprot.org/).The active compounds and potential targets of CGGE for treating IS were investigated based on network analysis. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  ≥ 0.18 . The pot) ≥ 0.18  were selhttps://www.ncbi.nlm.nih.gov/geo/, Series: GSE22255) database. Genes with p value <0.05 and |Fold change| > 1.2 were considered to be of significant differential expression and IS-related targets.The differential expressed genes of IS patients were obtained from Gene Expression Omnibus  was used for GO enrichment analysis, and plugin ClueGO of Cytoscape 3.9.0 software was used to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The biological process, cellular component, and molecular function analysis were selected for the bar charts. Pathways with significant changes of p < 0.05 were identified for further analysis.The Database for Annotation, Visualization, and Integrated Discovery  kits. The results were expressed as the means ± standard deviation.The protein concentration was calculated by using bicinchoninic acid (BCA) assay kit (KGI Biotechnology Co. Ltd. China). Identical quantities of protein samples were denatured by protein loading buffer, separated by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, then transferred to PVDF membranes . The PVDF membranes were sealed with 5% bovine serum albumin (BSA) at room temperature for one hour. The following antibodies were incubated at 4°C overnight: anti-CAMK4 , anti-p-CREB , anti-CREB1 , anti-NLRP3 , anti-ASC , anti-CASP1 . The stained blots were detected using an ECL reagent . Each blot was quantified using Bio-Rad Image Lab™ Version 3.0 software. All the experiments reported in this study were performed three times, and the results were reproducible.® SYBR® Green PCR Master Mix  with specific primers and expression of each sample in Light Cycler®480ⅡReal-time PCR Instrument , which internally normalized against Actb. The PCR primers for Camk4, Creb1, and Actb are as follows: Camk4 forward: 5′-CACAGAAATCAGCCTGGTT-3′ and reverse: 5′- ATCTGCTTCACAGCATCGC-3′; Creb1 forward: 5′- TTCTAGTGCCCAGCAACC-3′ and reverse: 5′-GAGGACGCCATAACAACTC-3′; Actb forward: 5′- CCACCATGTACCCAGGCATT-3′ and reverse: 5′- CGGACTCATCGTACTCCTGC-3′. Relative expression fold change was performed by using the 2−△△Ct method. Each experiment was repeated three times.According to the instructions, total RNA was extracted from the brain tissue using Trizol reagent . The concentration and purity of RNA were determined by a NanoDrop 2000 spectrophotometer . The absorbance ratio (A260/280) of all samples ranged from 1.8 to 2.0, and 2 μg of total RNA was reverse transcribed into cDNA using Prime Script RT Master Mix . RT-qPCR was determined using the QuantiFastp < 0.05 was considered statistically significant.The results were expressed as the mean ± standard deviation (SD). The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software . One-way analysis of variance (ANOVA) was used to compare all groups’ differences. The infarct volume of rats and neurological scores were determined after the acute treatment of the CGGE. As shown in p < 0.01), while the low dosage of CGGE group could only decrease the scores after 24 h (p < 0.05).Panax ginseng and 27 compounds of Ginkgo biloba were obtained. A total of 34 components were characterized after removing the compounds without targets and repetitions  with screening parameters of OB ≥ 30% and DL ≥ 0.18. Twenty-two compounds of etitions . The totThe network contained 169 nodes and 468 edges which indicated the compound-target interactions. The median degree of 34 chemical components is 9, indicating that compounds could interact with multiple targets. The information on the top 10 chemical components and targets is shown in The reliability of the test results can be significantly improved by directly selecting the human chip results and setting relevant screening conditions for analysis. The GEO database identified two hundred and thirty-nine IS-related targets (Series: GSE22255)..CGGE targets PPI network and IS-related targets PPI network were constructed. 6,275 CGGE target proteins and 5,259 IS-related target proteins were obtained . In orde.DAVID was used to perform GO analysis of the 288 identified candidate targets. GO of candidate targets was analyzed based on the domains “biological process,” “cellular component,” and “molecular function.” The top 10 terms are shown in , the mechanisms of CGGE against IS might be concerned with long-term potentiation, cAMP signaling pathway, neurotrophin signaling pathway, and NOD-like receptor signaling pathway.In order to further analyze the biological mechanisms involved with the 288 candidate targets, the ClueGO was used to perform the KEGG pathway analysis. As shown in n= 6). It was found that the Glu level was significantly higher in the MCE group than in the sham group (p < 0.01) . However, no significant differences in the GABA level were observed among all groups (p < 0.01). Treatment groups significantly reduced the Glu/GABA ratios compared to the MCE group (p < 0.01).The changes in Glu and GABA levels in the ipsilateral cortex were illustrated in  < 0.01) . The Tatl groups . The ratl groups  was founp < 0.01). CAMK4 and p-CREB expression changes were reversed following the treatments of Tat-NR2B9C and CGGE. The mRNA expression of Camk4 and Creb1 was decreased in the MCE group, whereas it increased with Tat-NR2B9C and the CGGE treatments (The effects of CGGE on the CAMK4/CREB are shown in eatments .n = 3). Results are shown in p < 0.01). Compared to the MCE group, CGGE significantly decreased the expression of NLRP3, ASC, and CASP1 proteins. In addition, CGGE exhibited concentration-dependent inhibition of the protein expression.To investigate the potential mechanism by which CGGE alleviates neuroinflammation, we examined the expression of NLRP3 inflammasome in the ipsilateral cortex  (2+/CaM activates CaM kinases (CaMKs) and CREB kinases from the surface membrane to the nucleus, which is important for synaptic potentiation, learning, and memory  . CRE count (CRE) . Ca2+/Cad memory . In varid memory . InteresCREB is a crucial transcription factor, and the inactivation of CREB leads to the inhibition of many CREs promoters, including neurotrophic factors and synaptic proteins. The activity of CREB is regulated by phosphorylation and dephosphorylation, and many protein kinases can phosphorylate CREB, such as protein kinase A (PKA) , mitogenInflammation plays a key role in the pathophysiology of ischemic stroke. NLRP3 inflammasome is the best-characterized inflammasome and one of the major contributors to neuroinflammation, which consists of NLRP3, ASC, and Caspase-1 . The KEGTaken as a whole, the present data suggested that the neuroprotective mechanism of CGGE against IS was related to excitotoxicity and neuroinflammation, which mainly through the long-term potentiation, cAMP signaling pathway, neurotrophin signaling pathway, and NOD-like receptor signaling pathway. However, there are still some limitations. Firstly, the rat model of cerebral ischemia was established by microsphere injection, which prevented an accurate assessment of the infarct sizes. Secondly, we only measured the content of amino acids 24 h after ischemic injury, while the change in amino acid level is a continuous process. More sampling points, especially during the early stage of ischemia, would help better understand the dynamic changes of the neurotransmitters and the related mechanisms. Therefore, further studies are expected to explore the deeper mechanism.in vivo experiments. The results showed that CGGE could improve neurological function via alleviating excitotoxicity and neuroinflammation. According to the network analysis, CALM1 might be the critical target of CGGE. Furthermore, the protective mechanisms of CGGE on ischemic stroke might involve long-term potentiation, cAMP signaling pathway, neurotrophin signaling pathway, and NOD-like receptor signaling pathway. The mechanisms by which CGGE exerts anti-inflammatory and anti-excitotoxicity effects involved the inhibition of NLRP3 inflammasome and upregulation of the CAMK4/CREB pathway.In conclusion, the underlying mechanisms of CGGE on IS were explored via the integration of network analysis and


To study the mechanism of osthole in GC based on network pharmacology and molecular docking.  The potential targets of osthole were predicted through the TCM System Pharmacology Analysis Platform, SwissTargetPrediction, and PharmMapper database. Targets related to gastric cancer were obtained through OMIM and GeneCard database. The online tool Venny 2.1.0 was used to screen GC and common target of osthole. The targets after the intersection of drugs and diseases were entered into the STRING database, and the protein interaction network was constructed, and the core targets with high correlation were screened out. The WebGestalt website was used to GO functional enrichment and KEGG pathway analysis and visualization with KOBAS website. The Cytoscape 3.8.2 was employed to draw the C-T-P-D (compound–target–pathway–disease) network visualization diagram. Finally, molecular docking validation was performed using PyMOL 2.5 and Discovery Studio Standalone.  Through prediction and screening, 108 corresponding targets were screened from osthole, and 173 targets were obtained after intersecting with gastric cancer targets. Among them, the top ten targets were the core targets of this study, including MAPK3, MAPK1, SRC, AKT1, HSP90AA1, RXRA, ESR1, RELA, MAPK14, and EGFR. The analysis of GO, KEGG, and PPI showed that the mechanism of action of osthole against GC may be closely related to the regulation of the PI3K signaling pathway. The results of molecular docking showed that osthole had a good affinity with MAPK3, which is a crucial part of the PI3K signaling pathway.  This study preliminarily revealed the targets and related pathways of osthole in the treatment of gastric cancer and provided a new idea for further exploration of osthole targeted prevention and treatment of gastric cancer. Gastric cancer (GC) is one of the common malignant tumors in clinic, which seriously threatens people's life and health. In most countries, gastric cancer occurs in the main part of the stomach (the corpus) and has high morbidity and mortality worldwide. The carcinogenesis of GC is a multistep and multistage development process involving Helicobacter pylori infection, geographic location, and other risk factors , 2. SurgOsthole is the main pharmacological active component of Chinese medicine Cnidium, which belongs to natural coumarin. Studies have shown that osthole has physiological and pharmacological effects such as osteogenesis, immune regulation, neuroprotection, and antioxidant functions –6. It alNetwork pharmacology provides a new network model of “multitarget, multipath, and multilink,” which has become an indispensable method for exploring the potential mechanism of medicines . It intehttp://tcmsp-e.com/), SwissTargetPrediction (http://www.swisstargetprediction.ch/), and PharmMapper (lilab-ecust.cn) and combined with literature to supplement the drug targets. On the other hand, use the GeneCards (https://www.genecards.org/) database and OMIM (https://omim.org/) database to obtain GC targets, select the species as “Homo sapiens,” and enter “Gastric Cancer” to retrieve GC-related genes. The online tool Venny 2.1.0 was used to analyze the common targets of GC and osthole and draw a Venn diagram.The compound structure of osthole (https://string-db.org/) platform for PPI network analysis, and the option was restricted to “Homo sapiens,” and the isolated targets were removed and screened with a high confidence level (0.95) to obtain the PPI network. Then, the PPI network was imported into Cytoscape 3.6.3; topological analysis was performed by the Cytohubba tool. According to the order of degree value, the most closely related targets (the top 10) were selected as the core targets of osthole anti-GC.The common genes obtained by mapping drugs and diseases are in STRING  and KOBAS (http://kobas.cbi.pku.edu.cn/) were used for GO enrichment analysis and KEGG pathway analysis. The core targets were imported, set the analyzed species as “Homo sapiens,” and the significance level P value < 0.05. These functions with a high significance level and a large frequency proportion will be considered as the potential biological functions of osthole against gastric cancer. The ImageGP (http://www.ehbio.com/ImageGP) website was employed to draw advanced bubble charts.The Webgestalt . Select the crystal structure of the target protein with high resolution (A) and complete structure from the PDB database (https://www.rcsb.org/) and download it. Use AutoDock Tools 1.5.6 to delete the water molecule of the target protein and add nonpolar sexual hydrogen and calculate Gasteiger charge; all flexible bonds are rotatable by default and save as pdbqt format file as docking ligand. Run AutoDock Vina 1.1.2 software for docking and calculate the docking score and use Pymol software for visualization.Molecular docking verification was performed on the top ten core targets of osthole against gastric cancer. Download compound mol2 format at Traditional Chinese Medicine Database@Taiwan  represented by SRC is a class of nonreceptor tyrosine kinases, and its role in signal transduction has been extensively studied . CurrentAs shown in the bubble chart, the PI3K/AKT signaling pathway and the MAPK signaling pathway accounted for a large number of targets. Phosphatidylinositol 3-kinase (PI3K) is an intracellular phosphatidylinositol kinase that upon activation produces the second messenger PIP3 at the plasma membrane and intracellular AKT. After binding to PIP3, it is transferred from the cytoplasm to the inside of the cell membrane for phosphorylation. As a key node in the pathway, AKT can affect the activation state of various downstream effector molecules. Studies have shown that in gastric cancer, osthole induces G2/M arrest and apoptosis by regulating the PI3K/Akt pathway. Osthole also exhibits proapoptotic properties in nude mouse retinoblastoma by inhibiting PI3K/Akt/mTOR signaling pathway activation , 23. At Molecular docking results showed that the binding energies of osthole and core targets were all less than −5 kJ/mol, which indicated that these core compounds had higher binding activities to receptor proteins, which further verified the results obtained by network pharmacology.In summary, this study used network pharmacology and molecular docking technology to preliminarily explore the multiple targets, biological functions, signaling pathways, and possible mechanisms of osthole in the treatment of gastric cancer and molecular docking to verify the results. The results show that osthole may act on MAPK3, MAPK1, AKT1, SRC, and other targets through PI3K/AKT, MAPK, and other signaling pathways, indicating that osthole has the potential to synergistically treat gastric cancer through multiple components, multiple targets, and multiple pathways. This study is mainly based on a web-based pharmacology and bioinformatics database. Therefore, there are some limitations. In the following studies, these data need to be further experimentally verified at the cellular, animal, and molecular levels to provide an experimental basis for the treatment of GC with osthole. It is hoped that osthole can become a new potential anti-GC drug.


The objective of this study was to determine the NF-kappaB pathway, hub genes, and transcription factors (TFs) in monocytes implicated in the progression of neurovascular-related sepsis-induced cardiomyopathy (SIC) as well as potential miRNAs with regulatory functions.: Sepsis-induced cardiomyopathy—and heart failure (HF)-related differentially expressed genes (DEGs) between SIC and HF groups were identified separately by differential analysis. In addition, DEGs and differentially expressed miRNAs (DEmiRNAs) in monocytes between sepsis and the HC group were identified. Then, common DEGs in SIC, HF, and monocyte groups were identified by intersection analysis. Based on the functional pathways enriched by these DEGs, genes related to the NF-kB-inducing kinase (NIK)/NF-kappaB signaling pathway were selected for further intersection analysis to obtain hub genes. These common DEGs, together with sepsis-related DEmiRNAs, were used to construct a molecular interplay network and to identify core TFs in the network.: A total of 153 upregulated genes and 25 downregulated genes were obtained from SIC-, HF-, and monocyte-related DEGs. Functional pathway analysis revealed that the upregulated genes were enriched in NF-κB signaling pathway. A total of eight genes associated with NF-κB signaling pathway were then further identified from the 178 DEGs. In combination with sepsis-related DEmiRNAs, HDAC7/ACTN4 was identified as a key transcriptional regulatory pair in the progression of SIC and in monocyte regulation. hsa-miR-23a-3p, hsa-miR-3175, and hsa-miR-23b-3p can regulate the progression of SIC through the regulation of HDAC7/ACTN4. Finally, gene set enrichment analysis (GSEA) suggested that HDAC7/ACTN4 may be associated with apoptosis in addition to the inflammatory response.: hsa-miR-23a-3p, hsa-miR-3175, and hsa-miR-23b-3p are involved in SIC progression by regulating NF-κB signaling signaling pathway-related HDAC7/ACTN4 in monocytes and cardiac tissue cells. These mechanisms may contribute to sepsis-induced neurovascular damage. Sepsis is a life-threatening organ dysfunction resulting from a dysregulated response of the organism to infection . CardiacResearchers have found that the degree of cardiac dysfunction is a major factor in predicting mortality and morbidity in sepsis . The groThe activation of NF-kappaB signaling pathways is closely associated with monocytes in SIC . MonocytmicroRNAs play a crucial regulatory role in the progression of various diseases –31. A stAdvancements in bioinformatics have enabled in-depth research into disease diagnosis and treatment from the perspective of big biological data –42. A laFirst, the keywords “Sepsis,” “Sepsis-induced cardiomyopathy (SIC),” “Septic cardiomyopathy (SCM),” and “Heart failure (HF)” were searched in the Gene Expression Omnibus (GEO) database. Datasets were filtered for bioinformatic analyses according to the following criteria: (1) data collected from human tissues; (2) single-cell RNA-sequencing (scRNA-seq) data; (3) sepsis and healthy control data; and (4) inclusion of at least 3 samples per group in the bulk RNA-seq transcriptome dataset. The GSE94717 , GSE1016As described in previous studies, RNA-seq data obtained from a total of 31,909 single cell from 5 samples were subjected to quality control using the “Seurat” package –55. The After integration of the scRNA-seq data, the “RunPCA” and “RunUMAP” functions were used to extract characteristics and reduce the dimensionality of single-cell transcripts. The “FindNeighbors” function was used to cluster the cells based on the default top 30 principal components (PCs), and the uniform manifold approximation and projection (UMAP) was used to visualize the cell clusters . Subsequp-Value < 0.05 was set as the threshold for DEG identification, and fold change values were used to identify upregulated and downregulated DEGs.At the level of scRNA-seq, monocytes were isolated to calculate monocyte-related DEGs in patients with sepsis (SP group) and healthy controls (HC group). The “FindMarker” function was used to identify monocyte-related DEGs, and the “ComBat” function was used to remove batch effects from different datasets before calculating DEmiRNAs. At the level of bulk-RNA analysis, the “limma” R package was used to identify SIC-related DEGs in the SIC and HC groups, HF-related DEGs in the HF and HC groups, and sepsis-related DEmiRNAs in the SP and HC groups at the individual level . p-Valuehttps://cran.r-project.org/web/packages/VennDiagram/index.html) was used to draw a Venn diagram to present the results of the intersection analysis.After identifying monocyte-, SIC-, and HF-related DEGs, intersection analysis was performed to identify common DEGs. Specifically, the analysis was performed separately for upregulated and downregulated DEGs, yielding co-upregulated or co-downregulated DEGs associated with monocytes, SICs, and HFs. Finally, the “VennDiagram” R package  and Gene Ontology (GO) analyses were performed using the “clusterProfiler” R package . GO termThe results of the functional pathway analysis suggested that the NF-kB-inducing kinase pathway (NIK)/NF-kappaB signaling pathway (GO: 0038061) was upregulated in the monocytes of patients with sepsis, SIC, and HF. Therefore, we further retrieved and collected a list of 144 genes associated with this pathway from the Molecular Signatures Database (MSigDB) –62. Genehttp://mirwalk.umm.uni-heidelberg.de/>), based on the identified sepsis-related DEmiRNAs, was used to identify predicted TF-target gene pairs (From TRRUST (v2), a list of TF and target genes with corresponding expression levels was downloaded and used to predict TF-target gene pairs in DEGs. Then, miRWalk (< ne pairs . Among tne pairs .The receiver operating characteristic (ROC) curve tool was used to assess the diagnostic capability of hub genes. In the horizontal and vertical coordinates of the curve, sensitivity was set on the y-axis, whereas “1-specificity”  was set on the x-axis. Thereafter, the area under the ROC curve (AUC) was calculated to quantify the diagnostic capability. As an indicator of diagnostic capability, the AUC value should be usually between 1.0 and 0.5, with a value closer to 1.0, indicating a more accurate diagnosis. In addition, the ROC curve was plotted using the “pROC” package .p-value < 0.05 was considered significant.In this study, statistical analyses were performed and graphical plots were created using R (version 4.0.2). All tests were two-sided, and a The distribution of key cell features, including feature counts, RNA counts, percentage of mitochondria (pMT), and percentage of hemoglobin (pHB), before quality control of the single-cell data, is shown in To examine the effects of optimal resolution on single-cell clustering, a range of resolution gradient values was selected . Based oThe differential expression analysis of DEGs associated with monocytes between the SP and HC groups yielded 4,429 upregulated and 380 downregulated DEGs , which wACTN4, DICER1, DLG1, HDAC7, NFAT5, PPP4C, TERF2IP, and TRIM44)  . The expp-values and fold change distributions of the differential analysis of HDAC7 and ACTN4 were shown in the volcano plot , which alter neurovascular-related HDAC7/ACTN4 signaling pathways linked to the NF-κB pathway. This study investigated the underlying mechanisms of neurovascular dysfunction associated with SIC and sepsis.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants or patients/participants legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.QL: software, validation, formal analysis, data curation, reviewing and editing, and writing—original draft. RL: conceptualization, methodology, supervision, funding acquisition, writing, reviewing, and editing. HM: methodology, conceptualization, project administration, funding acquisition, and software. EG: conceptualization, project administration and data curation. LY: software, data curation, and supervision. LJ and GF: data curation, writing, reviewing and editing, and data curation. BZ: methodology, supervision, funding acquisition, and data curation. All authors contributed to the article and approved the submitted version.The study was funded by Clinical Characteristic Discipline Construction Project of Shanghai Pudong New Area Health Commission (PWYts2021-17).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients with diabetic kidney disease (DKD) often have gastrointestinal dysfunction such as inflammatory bowel disease (IBD). This study aims to investigate the genetic mechanism leading to IBD in DKD patients through data mining and bioinformatics analysis.The disease-related genes of DKD and IBD were searched from the five databases of OMIM, GeneCards, PharmGkb, TTD, and DrugBank, and the intersection part of the two diseases were taken to obtain the risk genes of DKD complicated with IBD. A protein–protein interaction (PPI) network analysis was performed on risk genes, and three topological parameters of degree, betweenness, and closeness of nodes in the network were used to identify key risk genes. Finally, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on the risk genes to explore the related mechanism of DKD merging IBD.This study identified 495 risk genes for DKD complicated with IBD. After constructing a protein–protein interaction network and screening for three times, six key risk genes were obtained, including matrix metalloproteinase 2 (MMP2), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2), interleukin (IL)-18, IL-13, and C–C motif chemokine ligand 5 (CCL5). Based on GO enrichment analysis, we found that DKD genes complicated with IBD were associated with 3,646 biological processes such as inflammatory response regulation, 121 cellular components such as cytoplasmic vesicles, and 276 molecular functions such as G-protein-coupled receptor binding. Based on KEGG enrichment analysis, we found that the risk genes of DKD combined with IBD were associated with 181 pathways, such as the PI3K-Akt signaling pathway, advanced glycation end product–receptor for AGE (AGE-RAGE) signaling pathway and hypoxia-inducible factor (HIF)-1 signaling pathway.There is a genetic mechanism for the complication of IBD in patients with CKD. Oxidative stress, chronic inflammatory response, and immune dysfunction were possible mechanisms for DKD complicated with IBD. Diabetic kidney disease (DKD) is the most common cause of chronic kidney disease and end-stage renal disease worldwide, affecting approximately 30%–40% of people with diabetes . In receInflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract of unknown etiology. IBD has traditionally been viewed as a disease of the Western world. However, data over the past decade have shown that IBD has become a global disease with a dramatic increase in incidence and prevalence in the East . Due to In recent years, with the continuous development of molecular biotechnology, bioinformatics has played an increasingly important role in exploring the molecular mechanisms of human diseases through systematic analysis of available biomedical data . In thishttps://omim.org/) (https://www.genecards.org/), Pharmacogenomics Knowledge Base , Therapeutic Target Database , and DrugBank (https://www.drugbank.ca/). GeneCards is a comprehensive database that provides information on all predicted and annotated human genes, and genes with a correlation coefficient >10 are filtered as relevant genes  , GeneCarnt genes . PharmGKnt genes . TTD is nt genes . DrugBannt genes . The seaWe gathered the DKD- and IBD-related genes. The potential risk genes for DKD and IBD were identified from the above-shared genes.https://string-db.org/, version 11.0) is a database containing known and predicted PPI, which gathers information using bioinformatics strategies  was used to present the network. Lastly, Network Analyzer was used to calculate four topological parameters of each node in the network, including the degree, betweenness centrality, closeness centrality, and clustering coefficient  to clarify the possible biological mechanisms. KEGG (https://www.kegg.jp/) is a database for extracting biological information on functional classification, annotation, and enriched pathways of various genes. In this study, we used an R-package-Bioconductor clusterProfiler for GO and KEGG enrichment analysis. The R-package-Bioconductor clusterProfiler is widely used to automate biological term classification and enrichment analysis of gene clusters , cellular component (CC), and molecular function (MF), we used the Gene Ontology (GO) database , cell component (CC), and molecular function (MF) of 459 possible risk genes and six identified key risk genes, respectively, and the most relevant results that were chosen by p<0.05. These 181 pathways were involved in human diseases, pathophysiological mechanisms, and signaling pathways. The top 10 significantly enriched signaling pathways include the advanced glycation end product–receptor for AGE (AGE-RAGE) signaling pathway, Toll-like receptor signaling pathway, PI3K-Akt signaling pathway, hypoxia-inducible factor (HIF)-1 signaling pathway, tumor necrosis factor (TNF) signaling pathway, Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling pathway, IL-17 signaling pathway, T-cell receptor signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and Rap1 signaling pathway. The risk genes for DKD with IBD in the PI3K-Akt signaling pathway are depicted in To explore the underlying mechanisms of DKD complicated with IBD, KEGG pathway enrichment analysis was also performed on the risk genes and identified key risk genes, respectively, and the most relevant results are presented in Inflammatory bowel disease (IBD) is an autoimmune disease consisting of Crohn’s disease (CD), ulcerative colitis (UC), and indeterminate colitis. IBD is most common in Western countries, especially in North America and northern Europe; its prevalence is increasing year by year, and its cause is still unclear , 21. GenThrough data mining, we found that IBD and DKD have many related genes in common, and MMP2, HGF, FGF2, IL-18, IL-13, and CCL5 were identified as the core risk genes for DKD with IBD by PPI analysis. MMP2 is an enzyme with gelatinase activity. Overexpression of MMP2 in glomerulus and tubules can lead to kidney damage and fibrosis by inflammatory processes through various pathologies . MeanwhiAccording to the KEGG terms, the risk genes for DKD complicated with IBD were mainly associated with the PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and HIF-1 signaling pathway. Many studies have shown that stimulation of angiogenesis is an active participant in the establishment and maintenance of tissue inflammation in IBD , 48. TheOur study found that DKD and IBD share many common related genes and pathogenic mechanisms, oxidative stress, chronic inflammatory response, and immune dysfunction were possible mechanisms, and further research on these common targets for DKD complicated with IBD, especially the core targets, is important for the development of new effective drugs and the early diagnosis. Our study is based on the data mining and bioinformatics approach and has limitations due to the lack of experimental validation, mainly in the failure to validate the expression of the identified key risk genes in the blood of the patients with DKD combined with IBD and the changes in potential signaling pathways. Therefore, experiments on the key targets and related pathways of DKD combined with IBD are also needed to be conducted in the future.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/XZ performed the research, conducted the bioinformatics analysis, and wrote the first draft of the manuscript. HX, SF, and JY participated in data collection and analysis. YC and YJ participated in the project design and manuscript draft preparation and revision. All the authors have approved the final version of the manuscript.


Although modified Liu Jun Zi decoction (MLD) has favorable outcomes for chronic atrophic gastritis (CAG) in clinics, the identification of its active ingredients and the molecular mechanism of pharmacology are still unknown and need to be solved urgently. In the study, we screened 170 active components of MLD based on oral bioavailability ≥30% and drug-likeness ≥0.18 via the TCMSP platform. We further establish a dataset containing 315 CAG targets from PharmGkb, GeneCard, OMIM, DrugBank database, and Therapeutic Target database. Network pharmacology found that there are 110 active components of MLD and 26 potential targets for CAG in the “ingredient-target” network. The results of gene ontology analysis show that these targets are involved mainly in reactive oxygen species metabolic process, regulation of vasculature development, and T cell activation. KEGG pathways analysis indicates that these signaling pathways in the treatment of CAG include HIF-1 signaling pathway, neurodegeneration-multiple diseases pathway, MAPK signaling pathway, and PI3K-Akt signaling pathway. Finally, docking of the active component quercetin and clinical medicine Omeprazole with the core targets was carried out. We found that quercetin, a crucial active ingredient in MLD, has good binding activity with potential targets of CAG, and its molecular conformation is stable, which is better than the binding energy of Omeprazole. So, the active ingredients of MLD exhibit good potential drugs for the treatment of CAG. Chronic atrophic gastritis (CAG) is frequently an inflammatory disease of the digestive system , with epHelicobacter pylori infection is the most common cause of CAG, and more than 80% of CAG patients have detected positive for H. pylori sera. In addition, environmental factors, bile reflux, smoking and alcohol consumption, diet, and drugs are also a part of the risk factors for CAG. In the clinical practice, modern medical diagnosis of CAG is mainly based on the results of gastroscopy and biopsy. The red and white gastric mucosa can be seen in the stomach of CAG patients under endoscopy, and the white mucosa is the main one. Compared with normal gastric mucosa, the gastric mucosa vessels of CAG patients were exposed, the folds were flattened or disappeared, and there were small hyperplastic particles or larger nodules. Moreover, scattered patches of erosion and blood spots are seen in the patient's gastric mucosa.Clinically, CAG could be divided into two categories: autoimmune gastritis (AG) and multifocal atrophic gastritis (MAG) , 6. MAG So far, the etiology and specific pathogenesis of CAG are still not very clear, and there is a lack of effective drugs in clinical treatment. Clinically, a compound drug therapy is still dominant and has been extensively used for treating CAG. Moreover, the principle of individualization should be followed –10. Up tChronic atrophic gastritis has no definite disease name in ancient Chinese medicine literature. But, according to the fullness, epigastric pain, and other symptoms, it can be classified as “distention and fullness,” “stomach upset”, and “epigastric pain” in traditional Chinese medicine. In Huangdi Neijing, Shanghan Lun, Jingyue Quanshu, and other ancient medical books, the pathogenesis and syndrome differentiation of this disease are also clearly indicated. Modern scholars have systematically sorted and analyzed the literature on the diagnosis and treatment of CAG by famous veteran teran doctors of TCM and found that the most frequent understanding of its etiology and pathogenesis is spleen qi deficiency. On the whole, they believe that the pathogenesis of this disease is mainly based on deficiency in origin and excess in superficiality. For example, qi stagnation or phlegm dampness can be seen on the basis of spleen and stomach weakness. Syndrome differentiation and treatment is also quite effective. It has obvious effect in reducing symptoms, delaying, or reversing pathological changes and can provide important schemes and strategies for the prevention of gastric cancer.Panax Ginseng C. A. Mey (Renshen), Atractylodes macrocephala Koidz (Baizhu), Poria cocos (Schw.) Wolf (Fuling), Licorice (Gancao), Citrus Reticulata (Chenpi), and Arum ternatum Thunb (Banxia). The whole prescription has the effects of invigorating the spleen and stomach, reducing phlegm and regulating qi, and it was also employed to treat various digestive diseases such as gastritis, duodenitis, and inflammatory bowel diseases. In addition, this formula is also extensively used to treat gastric cancer and the side effects of radiotherapy and chemotherapy, which can improve the prognosis of patients. It is already in clinical trials in the United States and was approved for the European Union market by the German Food and Drug Administration in 2019. At present, Japan is at the forefront of the world in the research and marketization of this formula.Six Chinese herbs were composed in Liu Jun Zi decoction including Classical western medicine is a compound corresponding to a target to treat a disease. Its advantage is that the drug composition and pharmacological mechanism are clear. Traditional Chinese medicine, especially prescription drugs, often contains multiple active ingredients, which target multiple targets and work in multiple pathways. Traditional Chinese medicine prescription can achieve a comprehensive therapeutic effect, resulting in its complex pharmacology and difficulty to distinguish. With the continuous development of bioinformatics and system biology, the thinking mode of network pharmacology is more integrated and its predictive ability is more powerful in the research of traditional Chinese medicine.Codonopsis pargynoside, Atractylodes lactone II, hesperidin, and Pinellia ternata total alkaloids, and found that the content of active ingredients in Liu Jun Zi decoction increased significantly after processing, which could enhance the therapeutic effect. Gu et al. used liquid-mass spectrometry (LC-MS) to analyze the three active components in Liu Jun Zi decoction, including Atractylodes lactone III, quercetin, and fuling acid in Liu Jun Zi decoction, and found that only Atractylodes lactone III was detected in serum. Based on the integrated thinking of network pharmacology and strong predictive ability, Yang et al. analyzed 491 compounds contained in Sijunzi decoction (Codonopsis pilosula (Franch.) Nannf., Poria cocos (Schw.) Wolf, Atractylodes macrocephala Koidz, and licorice) and screened 123 active components corresponding to 78 targets of CAG. Further GO and KEGG analysis showed that Sijunzi decoction mainly regulates CAG by regulating cell inflammation, proliferation, apoptosis, and metabolism, which provides a basis for clarifying the mechanism of action of Sijunzi decoction in the treatment of CAG and conducting experimental verification. In view of the wide application of Liu Jun Zi decoction in the treatment of CAG, the mechanism of its active components needs to be clarified urgently. However, up to now, no systematic network pharmacological analysis of Liu Jun Zi decoction has been established for CAG.Hiroyuki et al.  found 32Coicis semen (Yiyiren) and Fritillariae thunbergii Bulbus (Zhebeimu). Coicis Semen is a kind of the medicine-food herb, known as the “King of Gramineae.” Esters, unsaturated fatty acids, sugars, triterpenoids, and lactams are the main active components of Coicis semen. In addition to the good effect of invigorating spleen and expeling dampness according to traditional Chinese medicine theory, the coix seed also has the functions of enhancing body immunity, regulating intestinal flora, regulating glucose and lipid metabolism, analgesia and anti-inflammatory, anticancer, and antioxidation activities. Moreover, clinical adverse reactions are less. Fritillaria thunbergia Bulbus is a traditional Chinese medicine for resolving phlegm and relieving cough, and it is said in the Mingyi Bielu that it can “treat the fullness under the heart.” According to the clinical experience of Mr. Xu Jingfan, a famous traditional Chinese medicine, Fritillaria thunbergii is often effective for some patients with difficult stomach diseases. Moreover, modern studies have found that there are alkaloids, polysaccharides, and total saponins in Fritillariae Thunbergii, among which alkaloids are the core active components. It has antitussive, expectorant, analgesia, antiulcer, antibacterial, antitumor and anti-inflammatory activities. Therefore, the addition of Coicis semen and Fritillaria Thunbergia Bulbus on the basis of Liu Jun Zi decoction is a reasonable compatibility based on the comprehensive consideration of the etiology and pathogenesis, clinical symptoms, pathological manifestations, and drug safety of CAG and has an ideal clinical effect. In this project, we analyzed the active components of modified Liu Jun Zi decoction. A series of analysis were conducted to explore the mechanism of modified Liu Jun Zi decoction in the treatment of CAG by multicomponent, multitarget and multichannel means, such as target analysis, network pharmacological analysis, pathway analysis of targeted regulation, analysis of CAG differential gene expression, molecular docking analysis of active components, compound cell assay, clinical treatment guides, and result analysis.In the study, the modified Liu Jun Zi decoction is based on Liu Jun Zi decoction with the addition of two traditional Chinese medicines, which are Panax ginseng C. A. Mey), Baizhu , Fuling (Poria Cocos(Schw.) Wolf), Gancao (Licorice), Chenpi (Citrus reticulata), Banxia (Arum ternatum Thunb), Zhebeimu (Fritillariae thunbrgii Bulbus), and Yiyiren (Coicis semen). The candidate ingredients of MLD were screened with oral bioavailability (OB) ≥ 30% and druglikelihood (DL) ≥ 0.18 [https://www.uniprot.org/uniprot).As shown in ) ≥ 0.18 , 13. Furhttps://www.omim.org), Therapeutic Target database (http://db.idrblab.net/ttd/), PharmGkb database (https://www.pharmgkb.org), DrugBank database (https://www.drugbank.ca), and GeneCard database (https://www.genecards.org). Then, the search target genes combined and deleted the duplicate genes to form a cluster of potential target information of CAG). Furthermore, the active ingredient targets of MLD and the disease targets of CAG were analyzed by drawing Venn diagrams with “venn” R package. The intersection target genes of MLD and CAG were collected as the common genes, which might be the potential targets of MLD for CAG treatment).Chronic atrophic gastritis-related target genes were searched and screened using atrophic gastritis as a keyword from five databases: OMIM database (The network of active ingredient of MLD and the disease targets of CAG were constructed by Cytoscape . CytoNCAhttps://string-db.org/cgi/input.pl). Common target genes of MLD and CAG were imported into the STRING database for constructing PPI network with a confidence level of 0.7 and to hide disconnected nodes [In this study, a lot of protein interactions were collected by STRING database . The sdf file of 2D compounds were converted to the mol2 files of 3D compounds with minimum free energy through Chem3D software. Subsequently, the crystal structure files of CAG target proteins were downloaded from RCSB PDB database (https://www.rcsb.org). The PyMOL-2.4.0 software was used to remove water molecules and small molecule ligands from the crystal structure of proteins.The molecular structure file of Omeprazole and core drug-like ingredients from network pharmacology were extracted from PubChem database , 7 ingredients from Atractylodes macrocephala Koidz (Baizhu), 7 ingredients from Fritillariae thunbergii Bulbus (Zhebeimu), and 13 ingredients from Arum ternatum Thunb (Banxia). As for Citrus reticulatae (Chenpi), there were 5 candidate ingredients in the specific Chinese herb. Poria cocos (Schw.) Wolf (Fuling) had 15 candidate ingredients. In addition, Licorice (Gancao) had 92 candidate ingredients and Coicis semen (Yiyiren) had 9 candidate ingredients. Furthermore, a total of 6286 target genes were in the 8 Chinese herbs of MLD. There were 748 in Panax ginseng C. A. Mey (Renshen), 774 in Atractylodes macrocephala Koidz (Baizhu), 110 in Fritillariae thunbergii Bulbus (Zhebeimu), 1302 in Arum ternatum Thunb (Banxia), 479 in Citrus reticulatae (Chenpi), 121 in Poria cocos (Schw.) Wolf (Fuling), 2506 in Licorice (Gancao) and 246 in Coicis semen (Yiyiren), respectively. Finally, we connected the candidate ingredients and MLD targets to obtain 2077 candidate ingredient targets of MLD  ≥ 30% and druglikelihood (DL) ≥ 0.18. Among them, 22 candidate ingredients were from We searched the target genes in five disease databases, PharmGkb, TTD, GeneCard, OMIM, and DrugBank, by the key words of “chronic atrophic gastritis,” as well as ultimately got 315 target genes of CAG  were analyzed, and the results showed that the BP were mainly involved in the metabolism and regulation of reactive oxygen species, the regulation of angiogenesis, and T cell activation, which were closely related to biological function. The CC were involve peroxisomes, microbodies, and nuclear transcription factor complexes. MF was mainly enriched in ligand receptor activation, phosphorylation, cytokine activation, etc. As shown in P < 0.05.The biological processes (BP), cell components (CC), and molecular function (MF) of 26 core targets , including BCL2, PRKCA, NOS2, STAT3, EGF, IL6R, ERBB2, and IFNG  and modified using Autodock 4.2 software  to remove ligands, add hydrogen, remove water, and optimize and patch amino acids. The protein preparation and binding pocket prediction were performed with Autodock. Subsequently, the receptor proteins and ligands were prepared with Chimera v1.6.2 prior to docking. The lower the binding energy of ligand and receptor, the more stable the conformation, and the greater the possibility of activity. The results reveal that quercetin possessed good binding activity with CCL2 (PDB ID: 1dok), CXCL8 (PDB ID : 5D14), EGFR (PDB ID : 1M17), PTGS2 (PDB ID: 5IKR), IL1B (PDB ID: 4i1b), TP53 (PDB ID: 3DCY), and IFNG (PDB ID: 1hig), which were potential targets of CAG, and the molecular conformational binding is stable. Its binding energy is better than that of Omeprazole, a western drug used in clinical treatment of CAG. The binding with potential targets is shown in We further analyzed the binding energy between MLD active compounds and CAG core targets by molecular docking . The co-MLD has the effect of invigorating spleen and regulating gastrointestinal diseases. Clinically, it is widely used to treat chronic atrophic gastritis. In addition, accumulated evidence has shown that MLD can reduce the side effects after radiotherapy and chemotherapy, which can improve the prognosis and prolong the survival of patients. Nevertheless, the identification of active ingredients and pharmacological molecular mechanism of MLD is less known, which needs to be solved urgently. Furthermore, the systematic network pharmacological analysis of modified Liu Jun Zi decoction has not been established up to now.α and MMP-9 [κB signaling pathways [In this study, the degree of quercetin, kaempferol, and naringenin was the highest in the ingredient-target network of MLD. Quercetin could inhibit inflammation and proteoglycan degradation through the downregulation of TNF-nd MMP-9 . Kaempfepathways , 21. In pathways . These fThe core targets of MLD for CAG treatment were CCL2, CXCL8, EGF, PTGS2, IL1B, TP53, and IFNG. The epidermal growth factor (EGF) is a potent promotor for carcinogenesis  via the α and an aryl hydrocarbon nuclear translocator HIF-1β. It has also been shown to regulate the expression of VEGF, EPO, and glycolytic enzymes [Helicobacter pylori infection could activate the PI3K/Akt pathway to promote the occurrence and development of CAG and tumors [The results of enrichment analysis revealed that the main pathways involved in the treatment process are HIF-1 signaling pathways, pathways of neurodegeneration-multiple disease, MAPK and PI3K-Akt signaling pathways, etc. HIF-1 is composed of an oxygen sensitive subunit HIF-1 enzymes . The Hua enzymes . Huang e enzymes . Moreoved tumors .In summary, we identified 110 drug components and 26 potential targets in MLD by using network pharmacological methods. It was preliminarily predicted that MLD could regulate the targets of CCL2, CXCL8, EGF, PTGS2, IL1B, TP53, and IFNG through quercetin, kaempferol, and naringenin. The regulation of HIF-1 signaling pathways, pathways of neurodegeneration-multiple disease, MAPK, and PI3K-Akt signaling pathways potentially inhibit the inflammatory response, immune function, and cell apoptosis to treat CAG. Molecular docking demonstrated that the binding energy of quercetin with CCL2, CXCL8, EGF, PTGS2, IL1B, TP53, and IFNG is better than that of Omeprazole, a western drug used in clinical treatment of CAG. However, the experiments still need to verify the finding results in vivo and in vitro, such as the phosphorylation and expression of target gene, as well as the activity regulation of target protein.