Mechanism of Artemisinic acid in regulating inflammation: Insights from network pharmacology and experimental validation

Mechanism of Artemisinic acid in regulating inflammation: Insights from network pharmacology and experimental validation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mechanism of Artemisinic acid in regulating inflammation: Insights from network pharmacology and experimental validation W Du, P Zhang, B Lan, M Wang, X L Mei, H Chen, S L Wu, L Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3926665/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Artemisinic acid is enriched in the well-known herbaceous plants Artemisia annua L. Due to the negtive anti-malarial activity of Artemisinic acid , it is only used as raw material for the synthesis of Artemisinin in industry. In fact, Artemisinic acid showed excellent effects on immune inflammation, but its mechanism is still unknown. In order to further develop and utilize Artemisinic acid, the reseach on its mechanism is conducted. In this study, network pharmacology methods were performed to analyze the potential targets of Artemisinic acid and the signaling pathways that regulate inflammation. The classic inflammation experimental methods were used, for example, Lipopolysaccharide (LPS) induces inflammation in rats, and the changes in general vital signs, hematology, cytokines, and key molecules of Interleukin-17(IL-17) signaling pathway were observed after Artemisinic acid remedy. In addition, the toxic effects of Artemisinic acid was alsoinvestigated.Research has demonstrated that artemisinin is a secure compound capable of inhibiting inflammation triggered by LPS. The inhibition of inflammation mechanism may be associated with the regulation of IL-17, ACT1 , Hsp90a , ERK , and COX-2 signaling within the IL-17 signaling pathway. Biological sciences/Drug discovery Biological sciences/Immunology Health sciences/Medical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Artemisinic acid is a crucial compound responsible for the production of artemisinin in Artemisia annua L. plants, with a remarkably high content 1 . However, it lacks anti-malaria pharmacological effects. As a result, in the industry, Artemisinic acid is solely utilized as a raw material for synthesizing Artemisinin, and uncatalyzed Artemisinic acid is regarded as an impurity 2 . Instantly, anti-inflammatory and immunomodulatory drugs have become the second largest category of drugs after antibiotics in clinical treatment 3 . In fact, as recently as 2009, Li Lanfang et al. discovered that Artemisinic acid can effectively alleviate yeast-induced fever in rats 4 . However, the mechanism behind this effect is still unknown. It is well-known that human fever is closely associated with the activation of the immune system under pathological conditions 5 . The most common cause of fever in the human body is infection with pathogens such as bacteria or viruses. When the human body gets infected with bacteria, the immune organs activate the innate and adaptive immune functions to promote immune defense 6 . Lipopolysaccharide (LPS), a product of pathogens, can enter the bloodstream and acts as a potent agonist that induces immune stress and adaptive immunity. Activation of the IL-17 signaling pathway serves as a basis for initiating adaptive immunity 7 . While the liver, bone marrow, and spleen all mount an immune response to pathogens, the spleen plays a central role in the blood defense system (BDS) 8 . Considering the antipyretic pharmacological effects of Artemisinic acid, it is hypothesized that this compound may regulate the immune stress caused by pathogens. In order to investigate this, we induced inflammation in rats using LPS and examined the immunoregulatory mechanism of Artemisinic acid in spleen tissue, specifically focusing on the IL-17 signaling pathway. Additionally, we evaluated the safety of Artemisinic acid. Further research on the immunomodulatory activity of artemisinin will contribute to the development of immunomodulatory drugs. Materials and Methods Putative of Drugs To conduct our computer simulation, we utilized five databases: PubChem (https://pubchem.ncbi.nlm.nih.gov/), FAF-Drugs4 (https://fafdrugs4.rpbs.univ-paris-diderot.fr/), SWISS (http://www.swisstargetprediction.ch/), GeneCards (https://www.genecards.org/), and STRING (http://string-db.org/). The detailed functions and usage of these databases have been extensively described in other academic papers 9, 10 . Simply put, we retrieved the Canonical SMILES of Artemisinic acid from the PubChem database and imported it into the FAF-Drugs4 database for simulation calculation of druggability. Additionally, we imported the Canonical SMILES into the SWISS database to predict the target of Artemisinic acid. To identify targets related to spleen immune stress, we conducted a systematic search of the GeneCards database using the search term 'LPS-induced inflammation', screening for targets with a score greater than or equal to 20.0. The predicted target of Artemisinic acid and the targets related to spleen immune stress were then cross-referenced in the STRING database for KEGG pathway enrichment analysis. Finally, based on the minimum FDR calculated by the database, we deduced the signal pathway regulated by Artemisinic acid. Experimental verification Drugs and Reagents . Artemisinic acidwere provided by Guangdong Wengjiang Pharmaceutical Co., Ltd.(Guangdong, China). Lipopolysaccharide（LPS, Escherichia coli O55:B55) were provided by Shanghai Aladdin Biochemical Technology Co., Ltd.(Shanghai, China).Interleukin-17 (IL-17), Cyclooxygenase-2 (COX-2), Interleukin-6(IL-6), Tumor necrosis factor-α (TNF-α), Cyclic adenosine monophosphate (cAMP), Prostaglandin E-2 (PGE2) detection kits were provided by Jiangsu enzyme Co., Ltd. (Jiangsu, China). Animals . SD rats were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). Rats were reared in a controlled environment with constant temperature (20～26 ℃), constant humidity (40～70%), alternating light and dark (12 h/12 h), and standard pellet feed and drinking water. All animal use was carried out in accordance with the animal guidelines and approved by the Animal Ethics Committee of Chongqing Academy of Chinese Materia Medica. L PS-induced inflammation in Rats and experimental design . In accordance with the method described in the literature, LPS was used to induce inflammation in rats 11 . The eligibility criteria for temperature monitoring in the General Vital Signs Monitor were followed to exclude unqualified rats. The rats were randomly divided into 4 groups (n=8): the normal control group (NC), the inflammation control group (IC), the Artemisinic acid group (AA), and the inflammation + Artemisinic acid group (IC+AA). IC and IC+AA groups were intraperitoneally injected with LPS at a dose of 100 µg/kg to induce immune stress in the spleen of rats, while NC and AA groups were intraperitoneally injected with pure normal saline in equal volumes. After four hours, the test substance and negative control substance were administered based on the respective groups. Specifically, the IC+AA group and the AA group were intraperitoneally injected with AA solution at a dose of 200 mg/kg, while the NC group and the IC group were intraperitoneally injected with an equal volume of normal saline. General vital signs monitor . Body temperature surveying: Prior to inducing inflammation (T0), the rats' body temperature was assessed twice through rectal temperature monitoring. Rats whose body temperature did not change more than 1 ℃ on both occasions were considered qualified rats and included in the experimental group. The average value of the two body temperature measurements was used as the baseline data. Four hours after inducing inflammation (T4), the body temperature of rats in each group was measured. An increase in body temperature of more than 1 ℃ in the IC and IC+AA groups was considered as an indication to start treatment. The rats' body temperature was measured at 0.5 hours (T4.5), 1 hour (T5), and 2 hours (T6) after treatment, and the difference in temperature compared to the baseline body temperature was calculated. Independent activity frequency assessment: Independent activity frequency was measured at T0, T4, and T6 using the independent activity frequency instrument (Shandong Scientific Instrument Co., Ltd, China). One rat from each group was placed in a detection box and allowed to adapt for 5 minutes before being tested for 10 minutes. The detection boxes for each group were rotated as per the plan (Figure 1). Routine blood examination . The blood was collected from the abdominal aorta as per the planned procedure. Subsequently, an automatic animal blood analyzer (Sysmex, Japan) was used to analyze the number of white blood cells (WBC), the number of neutrophils (NEUT#), and the proportion of neutrophils (NEUT%). Measurement of cytokine production and hypothalamic neurotransmitter . In the investigation of LPS-induced inflammation, researchers often focus on the inflammatory factors IL-17, COX-2, IL-6, TNF-α, as well as the hypothalamic transmitters cAMP and PGE2. The ELISA method was utilized to measure the levels of IL-17, COX-2, IL-6, and TNF-α in rat serum during the study. Hypothalamic homogenate was prepared using a 1:9 ratio of hypothalamic tissue to normal saline, and the levels of cAMP and PGE2 were assessed. All procedures were carried out following the instructions provided with the kit. RT-PCR analysis of key targets in IL-17 signaling pathway . Total RNA from spleen tissue was extracted using the trizol total RNA extraction kit (Shanghai yuanye Bio-Technology Co., Ltd, China). The extracted RNA was then reverse transcribed into cDNA using ALLMEEK (China). The primer sequences, synthesized by Bositai Biotech (Chongqing, China) Co., Ltd, were listed in the Table. The amplification of 40 cycles was performed using the standard procedure of three-step DBI PCR. Fluorescence quantitative PCR (Bio-Rad, USA) was used, and the relative expression of the target gene was calculated using the formula 2 - △△ Ct . Table. Sequences of qRT-PCR primers. Gene names Primer sequences IL-17 A Forward primer: 5´-TGCGTTTCCTCTATTGTCC-3´ Reverse primer: 5´-CGCCTTCTTTTCAGGGT-3´ IL-17 F Forward primer: 5´-CCAGGGCTGTTCTAATTCC-3´ Reverse primer: 5´-TTCGGTATGTGGCTTTGTC-3´ NF-kB Activator 1 ( ACT1 ) Forward primer: 5´-AAGGGACTGGATGTTGGAG-3´ Reverse primer: 5´-CCGTAGATTATGCCAGGGT-3´ Heat shock protein HSP 90-alpha ( Hsp90a ) Forward primer: 5´-TAAACTGGACTCGGGGAA-3´ Reverse primer: 5´-TTTGGTGCCTGACTTGG-3´ Extracellular regulated protein kinases ( ERK ) Forward primer: 5´-GGACTGTTGGCAGAAGGA-3´ Reverse primer: 5´-GGATAGAGCGGTCAAGCA-3´ β-actin Forward primer: 5´-ACAGGACAATGCGACTCC-3´ Reverse primer: 5´-TTCGGCAGTAAGCCAGAC-3´ GAPDH Forward primer: 5´-GTTGTGGCTGACATGCT-3´ Reverse primer: 5´-CCCAGGATGCCCTTTAGT-3´ Statistical Analysis . The data were presented as mean±SD. Repeated measures ANOVA was used to analyze the changes in body temperature and the frequency of independent activities, while one-way ANOVA was used for the remaining data. A significance level of P<0.05 was considered statistically significant. Results Artemisinic acid c ould be accepted as a drug, and its active effect may be related to the regulation of IL-17 signaling pathway The Canonical SMILES of Artemisinic acid provided by the PubChem database is CC1CCC(C2C1CCC(=C2)C)C(=C)C(=O)O. According to the FAF-Drugs4 database simulation calculation, Artemisinic acid has the potential to be accepted as a drug. The SWISS database simulation and calculation indicate that Artemisinic acid has 100 potential targets, including drug targets. From the GeneCards database, 157 spleen immune stress-related targets were obtained, which are considered disease targets. TNF, IL17A, CXCL8, NR3C1, and CD4 are common targets found in both drug targets and disease targets. KEGG pathway enrichment analysis revealed that these targets are enriched in 22 signal pathways, such as the IL-17 signaling pathway, rheumatoid arthritis, and cytokine receptor interaction. Notably, the IL-17 signaling pathway showed the lowest FDR value of 0.0000573, suggesting a potential association between this pathway and the drug activity of Artemisinic acid (Figure 2). Animal experiment results Artemisinic acid could improve the general vital signs: inhibit the rise of body temperature and promote the frequency of spontaneous activities In comparison to the NC, the injection of LPS noticeably elevated the body temperature of the rats (T4). When compared to the IC, it was observed that Artemisinic acid effectively suppressed the rise in body temperature of the rats, starting one hour after treatment (T5) and continuing until two hours after treatment (T6). Sole administration of Artemisinic acid did not have any significant impact on the body temperature. These findings are illustrated in Figure 3A. In comparison to the NC, the injection of LPS significantly decreased the frequency of independent activities in rats (T4). On the other hand, when compared to the IC, the frequency of independent activities in rats significantly increased 2 hours (T6) after treatment with Artemisinic acid. Administration of Artemisinic acid alone did not result in any change in the frequency of independent activities. These findings are shown in Figure 3B. Artemisinic acid could effectively inhibit the increase of WBC and NEUT in blood The results presented in Figure 4 show that the injection of LPS significantly increased the levels of WBC, NEUT%, and NEUT# in rats compared to the NC. However, when treated with Artemisinic acid, the increase in WBC, NEUT%, and NEUT# observed in the IC was effectively inhibited. It is worth noting that the administration of only Artemisinic acid did not cause any changes in the levels of WBC, NEUT%, or NEUT#. Artemisinic acid could reduce the production of inflammatory factors IL-17 and COX-2 in serum ，and c ould not regulate hypothalamic warming mediators The levels of IL-17, COX-2, and IL-6 in the serum of rats were significantly increased after the injection of LPS, as shown in Figure 5A-5D. However, there was no significant change in TNF-α. Following treatment with Artemisinic acid, the levels of IL-17 and COX-2 in the serum of rats were reduced compared to the IC group. It is important to note that administration of only Artemisinic acid did not cause any changes in IL-17, COX-2, IL-6, or TNF-α. The results presented in Figure 5E-5F demonstrate that the injection of LPS significantly increased the levels of cAMP and PGE2 in the rat hypothalamic homogenate when compared to the NC. However, treatment with Artemisinic acid (IC) did not significantly reduce the levels of cAMP and PGE2 in the rat hypothalamic homogenate. Furthermore, administration of only Artemisinic acid did not result in significant changes in the levels of cAMP and PGE2 in the rat hypothalamic homogenate. Artemisinic acid could regulate signals of IL-17 pathway The findings in Figure 6 demonstrate that the injection of LPS resulted in a significant increase in the expression of IL-17A , IL-17F , ACT1 , and Hsp90a in the spleen tissue of rats, while the expression of ERK showed a slight reduction with no significant change. However, treatment with Artemisinic acid led to a decrease in the levels of IL-17A , IL-17F , ACT1 , and Hsp90a in the spleen tissue compared to the IC, and there was a significant increase in the expression of ERK . It is important to note that administration of Artemisinic acid alone did not cause any changes in the expression of IL-17A , IL-17F , ACT1 , ERK , or Hsp90a . Discussion The application of computer-aided drug design methods has been shown to effectively reduce drug development time and screening costs 12 . Among the various methods used in computer-aided drug design, the network pharmacology method is widely recognized 13 . In our research, we utilized the network pharmacology method to simulate the potential of Artemisinic acid as a drug and predict its molecular targets. These predicted targets have the potential to treat LPS-induced inflammation, and it was observed that the targets involved in treating LPS-induced inflammation were enriched in the IL-17 signaling pathway. Our findings suggest that the activity of Artemisinic acid may be linked to the regulation of the IL-17 signaling pathway in the splenic organ (Figure 2). LPS is a reagent commonly used to induce immune stress in the spleen 14 . Injection of LPS in rats has been shown to cause changes in general vital signs, such as fever and decreased spontaneous activities 15, 16 . Body temperature and independent activities are often used as reference indicators to evaluate LPS-induced inflammation. In our model, we observed a significant increase in body temperature and a noticeable decrease in the frequency of independent activities after injecting rats with LPS, indicating successful mimicry of inflammation. Treatment with Artemisinic acid effectively inhibited the rise in body temperature and increased the frequency of independent activities in rats (Figure 3A and 3B). These findings suggest that Artemisinic acid may have the potential to counteract the adverse changes induced by LPS in body temperature and independent activities. Early detection and diagnosis of inflammation is crucial 17 . Hematological analysis shows an increase in white blood cell (WBC) count, as well as an increase in the NEUT# and NEUT% in the white blood cell classification, which aids in early detection and diagnosis 18, 19 . In this study, it was observed that Artemisinic acid effectively reduced blood WBC, NEUT#, and NEUT% (Figure 4), suggesting that Artemisinic acid may have potential in inhibiting the elevation of WBC, NEUT#, and NEUT% induced by LPS. IL-17, COX-2, IL-6, TNF-α, cAMP, and PGE2 are closely associated with the inflammation induced by LPS, exhibiting an overexpression trend in this experiment 20, 21 . Inflammatory factors play a crucial role in the inflammatory response, with IL-17, COX-2, IL-6, and TNF-α being secreted by immune organs 2 2 . When the immune system is under stress, the up-regulation of IL-17 and IL-6 in the blood is linked to the activation of immune cells in the spleen, leading to systemic inflammation 23, 24 . The overexpression of IL-17 and IL-6 triggers an increase in the expression of COX-2 and promotes the expression of PGE2 in the body 25 . In our experiments, we observed an overexpression of IL-17, COX-2, IL-6, cAMP, and PGE2. Treatment with artemisinic acid significantly reduced the levels of IL-17 and COX-2 (Figure 5). Notably, IL-17 serves as the initiating factor of the IL-17 signaling pathway, while COX-2 acts as the downstream factor of this pathway (https://www.kegg.jp/). These findings provide valuable insights into the regulatory role of artemisinic acid in the IL-17 signaling pathway. The IL-17 family is a significant cytokine family in humans, consisting of six related cytokines: IL-17A to IL-17F 26 . These cytokines have diverse and complex functions. While many IL-17 family members contribute to inflammation and play a role in defending against pathogens and immune-mediated diseases, they also play a part in maintaining mucosal integrity, responding to allergens, reducing inflammation, and regulating lymphocyte function. They can act in an autocrine manner and influence the function of other cytokines, including other IL-17 family members. LPS, a bacterial product, is a crucial pathogenic factor. The IL17 gene is primarily produced by Th17 cells but can also be expressed by neutrophils. It exerts an immunoprotective effect against fungi and bacteria and promotes the body's immune response 27, 28 . Therefore, overexpression of IL-17 and activation of the IL-17 signaling pathway serve as protective mechanisms against LPS-induced damage to the body. The functions of IL-17A and IL-17F are well understood. IL-17A and IL-17F have the ability to form homo- and heterodimers, which bind to the IL-17 receptor and activate downstream signaling pathways. This activation leads to the release of pro-inflammatory cytokines 29 . The recruitment of IL-17A-IL-17F stimulates immune cell recruitment of ACT1-Hsp90a, resulting in an increase in the release of cellular inflammatory factors 30 and promoting the secretion of COX-2 and IL-6. When the body is infected and the body temperature rises significantly, ACT1 and Hsp90a levels also rise significantly 5 . However, in the IL-17 signaling pathway, ERK inhibits the secretion of cytokines COX-2 and IL-6. Therefore, ERK may exhibit insignificant changes or low expression 31 . In rats injected with LPS, the expression of IL-17A, IL-17F, ACT1, and Hsp90a in the spleen tissue visibly increased. After treatment with Artemisinic acid, the expression of IL-17A, IL-17F, ACT1, and Hsp90a markedly reduced, while the expression of ERK increased (Figure 6). This suggests that the activity of Artemisinic Acid may be related to the regulation of IL-17A, IL-17F, ACT1, Hsp90a, and ERK in the IL-17 signaling pathway. The effects of administering Artemisinic acid were studied only on rats. No effects were found on body temperature, voluntary activity, hematological WBC counts, NEUT# and NEUT% in white blood cell classification, IL-17, COX-2, IL-6, TNF-α, cAMP, PGE2, ACT1, Hsp90a, or ERK (Figure 2-6). These results indicate that Artemisinic acid is not toxic, and all the significant changes observed in the experiment can be attributed to the pharmacological activity of Artemisinic acid. Conclusions This study utilized network pharmacology to simulate the activity of Artemisinic acid, suggesting a potential connection between its mechanism and the regulation of the IL-17 signaling pathway. Subsequent experimental verification confirmed that Artemisinic acid effectively improved immune stress symptoms in rats, reduced inflammation, and regulated IL-17, ACT1, Hsp90a, ERK, and COX-2 signals, thereby modulating the IL-17 signaling pathway. Importantly, preliminary results indicated the safety of Artemisinic acid application. In conclusion, these findings demonstrate that Artemisinic acid is a safe compound with immunomodulatory activity, potentially achieved through the regulation of the IL-17 signaling pathway. Abbreviations LPS Lipopolysaccharide IL-17 Interleukin-17 WBC White blood cells NEUT# Number of neutrophils NEUT% Proportion of neutrophils COX-2 Cyclooxygenase-2 IL-6 Interleukin-6 TNF-α Tumor necrosis factor-α cAMP Cyclic adenosine monophosphate PGE2 Prostaglandin E-2 ACT1 NF-kB Activator 1 Hsp90a Heat shock protein HSP 90-alpha ERK Extracellular regulated protein kinases Declarations Data Availability All data that support the findings of this study are included in this published article. Conflicts of Interest The authors report no conflicts of interest in this work. Funding Statement This study was supported by Chongqing basic scientific research business fee (jbky20200014) ,Chongqing General Research and Development Project (cstc2019jxjl-jbky10005) and Chongqing Municipal Health Commission Chongqing Key Discipline Construction Fund of Traditional Chinese Medicine(Pharmacology and Toxicology of Traditional Chinese Medicine). ACKNOWLEDGMENTS We thank RuxiaTu and Chonggang Huang, members of the Institute of pharmacology and toxicology of Traditional Chinese Medicine, Chongqing Academy of Chinese Materia Medica. References X Zhang, Y Zhao, L Guo, Z Qiu, L Huang, X Qu et al. Differences in chemical constituents of Artemisia annua L from different geographical regions in China. Plos One . 12(9): e0183047; 10.1371/journal.pone.0183047 (2017). BX Zeng, MD Yao, Y Wang, WH Xiao, YJ Yuan. Metabolic Engineering of Saccharomyces cerevisiae for Enhanced Dihydroartemisinic Acid Production. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3926665","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":272573379,"identity":"05545686-7b1b-4a18-8316-594151ed5fe2","order_by":0,"name":"W Du","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqklEQVRIiWNgGAWjYDCCA0CcUEG6ljMka2FsI0UH340cwwcP59nJ9refTt3AUGMTTVCL5I0cY4PEbcnGM87kbrvBcCwtt4GQFoMbudskErcdSGy4wbvtBmPDYaK0bP+ROOdA4nxStGxjSGw4kLiBaC2SZ95/lkg4lmy8EeSXBGL8wnc8LfHjjxo72XnHz2678aHGhrAWGGAEq0wgVjlCyygYBaNgFIwCbAAAA75KO8raEwQAAAAASUVORK5CYII=","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":true,"prefix":"","firstName":"W","middleName":"","lastName":"Du","suffix":""},{"id":272573380,"identity":"6edd9d5d-a8ea-4cc8-8c49-50d6e865167e","order_by":1,"name":"P Zhang","email":"","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"P","middleName":"","lastName":"Zhang","suffix":""},{"id":272573381,"identity":"4ad500ac-8806-4a47-9fc8-b285456ffb9d","order_by":2,"name":"B Lan","email":"","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"B","middleName":"","lastName":"Lan","suffix":""},{"id":272573382,"identity":"41ce1a1e-8f69-44b0-a494-ec8b0273d526","order_by":3,"name":"M Wang","email":"","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"M","middleName":"","lastName":"Wang","suffix":""},{"id":272573383,"identity":"be9fbc9c-a6c1-4509-97c1-6bd9c2761069","order_by":4,"name":"X L Mei","email":"","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"X","middleName":"L","lastName":"Mei","suffix":""},{"id":272573384,"identity":"b45ee838-82a5-4e0b-9e70-88f6a8d79ce6","order_by":5,"name":"H Chen","email":"","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"H","middleName":"","lastName":"Chen","suffix":""},{"id":272573385,"identity":"509dba21-402c-4340-ae3f-c062fd6bc84c","order_by":6,"name":"S L Wu","email":"","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"S","middleName":"L","lastName":"Wu","suffix":""},{"id":272573386,"identity":"1cb61c0b-4b61-49e7-ac94-f5f9bbe7f378","order_by":7,"name":"L Zhang","email":"","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"L","middleName":"","lastName":"Zhang","suffix":""},{"id":272573387,"identity":"d86c4346-0332-42e7-ad5a-979f7cceb9b4","order_by":8,"name":"Henghua H Li","email":"","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"Henghua","middleName":"H","lastName":"Li","suffix":""},{"id":272573388,"identity":"76b88608-c527-46c2-a414-bc7e32a17507","order_by":9,"name":"Xiaoxian X Song","email":"","orcid":"","institution":"Chongqing Academy of Chinese Materia Medica","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxian","middleName":"X","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2024-02-04 07:29:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3926665/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3926665/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104400590,"identity":"0d9f52a2-4c4a-42cf-8e9b-3fb1b324821c","added_by":"auto","created_at":"2026-03-11 12:10:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":123037,"visible":true,"origin":"","legend":"\u003cp\u003eDetection box rotation plan. All rats were monitored for independent activity frequency according to the rotation plan shown in the figure.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3926665/v1/caed481a0266b4f90b099066.png"},{"id":103812197,"identity":"21624fe2-afae-4b1a-a271-4c5feddaa0f6","added_by":"auto","created_at":"2026-03-03 08:29:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32049,"visible":true,"origin":"","legend":"\u003cp\u003eComputer simulation of Artemisinic acid regulating the potential signal pathways of inflammation induced by LPS. L-17 signaling pathway had the lowest FDR value, which may be related to the drug activity of Artemisinic acid.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3926665/v1/27ac14dca4db2de4d0dab381.png"},{"id":103812195,"identity":"ea6a5d97-b1f6-4dc2-b4d5-a5f99af33a44","added_by":"auto","created_at":"2026-03-03 08:29:48","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107266,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Artemisinic acid on general vital signs in normal rats and rats with inflammation induced by LPS. (A) Curve of body temperature change. (B) Independent activities frequency. The data were expressed as mean ± SD (n=8). **\u003cem\u003eP＜\u003c/em\u003e0.01 vs NC; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP＜\u003c/em\u003e0.05\u003cem\u003e vs\u003c/em\u003e IC.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3926665/v1/42cc5435a35876ef70a02721.jpeg"},{"id":103812199,"identity":"22e3774c-2a49-49f6-ba0a-8d10ea641c8b","added_by":"auto","created_at":"2026-03-03 08:29:48","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53877,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Artemisinic acid on the white blood cells and neutrophils in the blood of normal rats and rats with inflammation induced by LPS. (A) WBCcount. (B) The NEUT%. (C) NEUT count. The data were expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e=8). \u003cem\u003e*P \u003c/em\u003e\u0026lt;0.05 \u003cem\u003evs\u003c/em\u003e NC; \u003cem\u003e# P \u003c/em\u003e\u0026lt;0.05 \u003cem\u003evs\u003c/em\u003e IC.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3926665/v1/5506eaf91022375ae86c586e.jpeg"},{"id":104400320,"identity":"8b76b37c-37ce-4699-b74f-fff649c3fbff","added_by":"auto","created_at":"2026-03-11 12:09:36","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":127823,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Artemisinic acid on cytokines in serum and neurotransmitters in hypothalamus of normal rats and rats with inflammation induced by LPS. (A) IL-17 level in serum. (B) COX-2 level in serum. (C) The level of TNF-α in serum. (D) IL-6 level in serum. (E) cAMP level in the hypothalamus. (F) PGE2 level in the hypothalamus. The data were expressed as mean ± SD (\u003cem\u003en\u003c/em\u003e=8). \u003cem\u003e*P \u003c/em\u003e\u0026lt;0.05 \u003cem\u003evs\u003c/em\u003e NC ; \u003csup\u003e\u003cem\u003e# \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05 \u003cem\u003evs \u003c/em\u003eIC .\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3926665/v1/67ea8d89db8838fd8ed96be5.jpeg"},{"id":104400978,"identity":"a9617284-4a84-4e75-91c2-9eaf6668e593","added_by":"auto","created_at":"2026-03-11 12:11:37","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":97686,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Artemisinic acid on the key targets of IL-17 signaling pathway in spleen tissue in normal rats and rats with LPS-induced inflammation. (A) Relative expression level of \u003cem\u003eACT1\u003c/em\u003e. (B) Relative expression level of \u003cem\u003eERK\u003c/em\u003e. (C) Relative expression level of \u003cem\u003eHsp90a\u003c/em\u003e. The data were expressed as mean±SD (\u003cem\u003en\u003c/em\u003e=6～8). \u003cem\u003e*P \u003c/em\u003e\u0026lt;0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt;0.01 \u003cem\u003evs\u003c/em\u003e NC; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt;0.05 \u003cem\u003evs \u003c/em\u003eIC.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3926665/v1/12682495a7d8cbf49301998f.jpeg"},{"id":104410597,"identity":"d3e3f8e9-0e1b-4b4a-ae9f-b00f6972b8ac","added_by":"auto","created_at":"2026-03-11 12:53:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1320653,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3926665/v1/eb3df442-3436-48f4-bd71-95a992f2075f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanism of Artemisinic acid in regulating inflammation: Insights from network pharmacology and experimental validation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eArtemisinic acid is a crucial compound responsible for the production of artemisinin in Artemisia annua L. plants, with a remarkably high content \u003csup\u003e1\u003c/sup\u003e. However, it lacks anti-malaria pharmacological effects. As a result, in the industry, Artemisinic acid is solely utilized as a raw material for synthesizing Artemisinin, and uncatalyzed Artemisinic acid is regarded as an impurity\u0026nbsp;\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eInstantly, anti-inflammatory and immunomodulatory drugs have become the second largest category of drugs after antibiotics in clinical treatment\u0026nbsp;\u003csup\u003e3\u003c/sup\u003e. In fact, as recently as 2009, Li Lanfang et al. discovered that Artemisinic acid can effectively alleviate yeast-induced fever in rats\u0026nbsp;\u003csup\u003e4\u003c/sup\u003e. However, the mechanism behind this effect is still unknown.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is well-known that human fever is closely associated with the activation of the immune system under pathological conditions\u0026nbsp;\u003csup\u003e5\u003c/sup\u003e. The most common cause of fever in the human body is infection with pathogens such as bacteria or viruses. When the human body gets infected with bacteria, the immune organs activate the innate and adaptive immune functions to promote immune defense\u0026nbsp;\u003csup\u003e6\u003c/sup\u003e. Lipopolysaccharide (LPS), a product of pathogens, can enter the bloodstream and acts as a potent agonist that induces immune stress and adaptive immunity. Activation of the IL-17 signaling pathway serves as a basis for initiating adaptive immunity\u0026nbsp;\u003csup\u003e7\u003c/sup\u003e. While the liver, bone marrow, and spleen all mount an immune response to pathogens, the spleen plays a central role in the blood defense system (BDS)\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eConsidering the antipyretic pharmacological effects of Artemisinic acid, it is hypothesized that this compound may regulate the immune stress caused by pathogens. In order to investigate this, we induced inflammation in rats using LPS and examined the immunoregulatory mechanism of Artemisinic acid in spleen tissue, specifically focusing on the IL-17 signaling pathway. Additionally, we evaluated the safety of Artemisinic acid. Further research on the immunomodulatory activity of artemisinin will contribute to the development of immunomodulatory drugs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePutative of Drugs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo conduct our computer simulation, we utilized five databases: PubChem (https://pubchem.ncbi.nlm.nih.gov/), FAF-Drugs4 (https://fafdrugs4.rpbs.univ-paris-diderot.fr/), SWISS (http://www.swisstargetprediction.ch/), GeneCards (https://www.genecards.org/), and STRING (http://string-db.org/). The detailed functions and usage of these databases have been extensively described in other academic papers \u003csup\u003e9, 10\u003c/sup\u003e. Simply put, we retrieved the Canonical SMILES of Artemisinic acid from the PubChem database and imported it into the FAF-Drugs4 database for simulation calculation of druggability. Additionally, we imported the Canonical SMILES into the SWISS database to predict the target of Artemisinic acid. To identify targets related to spleen immune stress, we conducted a systematic search of the GeneCards database using the search term \u0026apos;LPS-induced inflammation\u0026apos;, screening for targets with a score greater than or equal to 20.0. The predicted target of Artemisinic acid and the targets related to spleen immune stress were then cross-referenced in the STRING database for KEGG pathway enrichment analysis. Finally, based on the minimum FDR calculated by the database, we deduced the signal pathway regulated by Artemisinic acid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Experimental verification\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrugs and Reagents\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e Artemisinic\u0026nbsp;acidwere provided by Guangdong Wengjiang Pharmaceutical Co., Ltd.(Guangdong, China). Lipopolysaccharide（LPS, Escherichia coli O55:B55) were provided by Shanghai Aladdin Biochemical Technology Co., Ltd.(Shanghai, China).Interleukin-17 (IL-17), Cyclooxygenase-2 (COX-2), Interleukin-6(IL-6), Tumor necrosis factor-\u0026alpha; (TNF-\u0026alpha;), Cyclic adenosine monophosphate (cAMP), Prostaglandin E-2 (PGE2) detection kits were provided by Jiangsu enzyme Co., Ltd. (Jiangsu, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSD rats were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). Rats\u0026nbsp;were\u0026nbsp;reared in a controlled environment with constant temperature (20～26 ℃), constant humidity (40～70%), alternating light and dark (12 h/12 h), and standard pellet feed and drinking water. All animal use was carried out in accordance with the animal guidelines and approved by the Animal Ethics Committee of\u0026nbsp;Chongqing Academy of Chinese Materia Medica.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL\u003c/strong\u003e\u003cstrong\u003ePS-induced inflammation in Rats and experimental design\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eIn accordance with the method described in the literature, LPS was used to induce inflammation in rats\u0026nbsp;\u003csup\u003e11\u003c/sup\u003e. The eligibility criteria for temperature monitoring in the General Vital Signs Monitor were followed to exclude unqualified rats. The rats were randomly divided into 4 groups (n=8): the normal control group (NC), the inflammation control group (IC), the Artemisinic acid group (AA), and the inflammation + Artemisinic acid group (IC+AA). IC and IC+AA groups were intraperitoneally injected with LPS at a dose of 100 \u0026micro;g/kg to induce immune stress in the spleen of rats, while NC and AA groups were intraperitoneally injected with pure normal saline in equal volumes. After four hours, the test substance and negative control substance were administered based on the respective groups. Specifically, the IC+AA group and the AA group were intraperitoneally injected with AA solution at a dose of 200 mg/kg, while the NC group and the IC group were intraperitoneally injected with an equal volume of normal saline.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral vital signs monitor\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBody temperature surveying: Prior to inducing inflammation (T0), the rats\u0026apos; body temperature was assessed twice through rectal temperature monitoring. Rats whose body temperature did not change more than 1 ℃ on both occasions were considered qualified rats and included in the experimental group. The average value of the two body temperature measurements was used as the baseline data. Four hours after inducing inflammation (T4), the body temperature of rats in each group was measured. An increase in body temperature of more than 1 ℃ in the IC and IC+AA groups was considered as an indication to start treatment. The rats\u0026apos; body temperature was measured at 0.5 hours (T4.5), 1 hour (T5), and 2 hours (T6) after treatment, and the difference in temperature compared to the baseline body temperature was calculated.\u003c/p\u003e\n\u003cp\u003eIndependent activity frequency assessment: Independent activity frequency was measured at T0, T4, and T6 using the independent activity frequency instrument (Shandong Scientific Instrument Co., Ltd, China). One rat from each group was placed in a detection box and allowed to adapt for 5 minutes before being tested for 10 minutes. The detection boxes for each group were rotated as per the plan (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRoutine\u0026nbsp;blood\u0026nbsp;examination\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eThe blood was collected from the abdominal aorta as per the planned procedure. Subsequently, an automatic animal blood analyzer (Sysmex, Japan) was used to analyze the number of white blood cells (WBC), the number of neutrophils (NEUT#), and the proportion of neutrophils (NEUT%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of cytokine production and hypothalamic neurotransmitter\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eIn the investigation of LPS-induced inflammation, researchers often focus on the inflammatory factors IL-17, COX-2, IL-6, TNF-\u0026alpha;, as well as the hypothalamic transmitters cAMP and PGE2. The ELISA method was utilized to measure the levels of IL-17, COX-2, IL-6, and TNF-\u0026alpha; in rat serum during the study. Hypothalamic homogenate was prepared using a 1:9 ratio of hypothalamic tissue to normal saline, and the levels of cAMP and PGE2 were assessed. All procedures were carried out following the instructions provided with the kit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-PCR analysis of key targets in IL-17 signaling pathway\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eTotal RNA from spleen tissue was extracted using the trizol total RNA extraction kit (Shanghai yuanye Bio-Technology Co., Ltd, China). The extracted RNA was then reverse transcribed into cDNA using ALLMEEK (China). The primer sequences, synthesized by Bositai Biotech (Chongqing, China) Co., Ltd, were listed in the Table. The amplification of 40 cycles was performed using the standard procedure of three-step DBI PCR. Fluorescence quantitative PCR (Bio-Rad, USA) was used, and the relative expression of the target gene was calculated using the formula 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTable. Sequences of qRT-PCR primers.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 237px;\"\u003e\n \u003cp\u003eGene names\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003ePrimer sequences\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 237px;\"\u003e\n \u003cp\u003e\u003cem\u003eIL-17\u003c/em\u003e\u003cem\u003eA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eForward primer: 5\u0026acute;-TGCGTTTCCTCTATTGTCC-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eReverse primer: 5\u0026acute;-CGCCTTCTTTTCAGGGT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 237px;\"\u003e\n \u003cp\u003e\u003cem\u003eIL-17\u003c/em\u003e\u003cem\u003eF\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eForward primer: 5\u0026acute;-CCAGGGCTGTTCTAATTCC-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eReverse primer: 5\u0026acute;-TTCGGTATGTGGCTTTGTC-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 237px;\"\u003e\n \u003cp\u003e\u003cem\u003eNF-kB Activator 1\u0026nbsp;\u003c/em\u003e(\u003cem\u003eACT1\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eForward primer: 5\u0026acute;-AAGGGACTGGATGTTGGAG-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eReverse primer: 5\u0026acute;-CCGTAGATTATGCCAGGGT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 237px;\"\u003e\n \u003cp\u003e\u003cem\u003eHeat shock protein HSP 90-alpha\u0026nbsp;\u003c/em\u003e(\u003cem\u003eHsp90a\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eForward primer: 5\u0026acute;-TAAACTGGACTCGGGGAA-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eReverse primer: 5\u0026acute;-TTTGGTGCCTGACTTGG-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 237px;\"\u003e\n \u003cp\u003e\u003cem\u003eExtracellular regulated protein kinases\u003c/em\u003e(\u003cem\u003eERK\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eForward primer: 5\u0026acute;-GGACTGTTGGCAGAAGGA-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eReverse primer: 5\u0026acute;-GGATAGAGCGGTCAAGCA-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 237px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026beta;-actin\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eForward primer: 5\u0026acute;-ACAGGACAATGCGACTCC-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eReverse primer: 5\u0026acute;-TTCGGCAGTAAGCCAGAC-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 237px;\"\u003e\n \u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eForward primer: 5\u0026acute;-GTTGTGGCTGACATGCT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 379px;\"\u003e\n \u003cp\u003eReverse primer: 5\u0026acute;-CCCAGGATGCCCTTTAGT-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e The data were presented as mean\u0026plusmn;SD. Repeated measures ANOVA was used to analyze the changes in body temperature and the frequency of independent activities, while one-way ANOVA was used for the remaining data. A significance level of \u003cem\u003eP\u0026lt;0.05\u003c/em\u003e was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eArtemisinic acid c\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eould be accepted as a drug, and its active effect may be related to the regulation of IL-17 signaling pathway\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Canonical SMILES of Artemisinic acid provided by the PubChem database is CC1CCC(C2C1CCC(=C2)C)C(=C)C(=O)O. According to the FAF-Drugs4 database simulation calculation, Artemisinic acid has the potential to be accepted as a drug. The SWISS database simulation and calculation indicate that Artemisinic acid has 100 potential targets, including drug targets. From the GeneCards database, 157 spleen immune stress-related targets were obtained, which are considered disease targets. TNF, IL17A, CXCL8, NR3C1, and CD4 are common targets found in both drug targets and disease targets. KEGG pathway enrichment analysis revealed that these targets are enriched in 22 signal pathways, such as the IL-17 signaling pathway, rheumatoid arthritis, and cytokine receptor interaction. Notably, the IL-17 signaling pathway showed the lowest FDR value of 0.0000573, suggesting a potential association between this pathway and the drug activity of Artemisinic acid (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAnimal experiment results\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eArtemisinic acid could improve the general vital signs: inhibit the rise of body temperature and promote\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe frequency of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003espontaneous activities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn comparison to the NC, the injection of LPS noticeably elevated the body temperature of the rats (T4). When compared to the IC, it was observed that Artemisinic acid effectively suppressed the rise in body temperature of the rats, starting one hour after treatment (T5) and continuing until two hours after treatment (T6). Sole administration of Artemisinic acid did not have any significant impact on the body temperature. These findings are illustrated in Figure 3A.\u003c/p\u003e\n\u003cp\u003eIn comparison to the NC, the injection of LPS significantly decreased the frequency of independent activities in rats (T4). On the other hand, when compared to the IC, the frequency of independent activities in rats significantly increased 2 hours (T6) after treatment with Artemisinic acid. Administration of Artemisinic acid alone did not result in any change in the frequency of independent activities. These findings are shown in Figure 3B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eArtemisinic acid could effectively inhibit the increase of WBC and NEUT in blood\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results presented in Figure 4 show that the injection of LPS significantly increased the levels of WBC, NEUT%, and NEUT# in rats compared to the NC. However, when treated with Artemisinic acid, the increase in WBC, NEUT%, and NEUT# observed in the IC was effectively inhibited. It is worth noting that the administration of only Artemisinic acid did not cause any changes in the levels of WBC, NEUT%, or NEUT#.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eArtemisinic acid\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecould reduce the production of inflammatory factors IL-17 and COX-2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ein serum\u003c/strong\u003e\u003cstrong\u003e，and c\u003c/strong\u003e\u003cstrong\u003eould not regulate hypothalamic warming mediators\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe levels of IL-17, COX-2, and IL-6 in the serum of rats were significantly increased after the injection of LPS, as shown in Figure 5A-5D. However, there was no significant change in TNF-\u0026alpha;. Following treatment with Artemisinic acid, the levels of IL-17 and COX-2 in the serum of rats were reduced compared to the IC group. It is important to note that administration of only Artemisinic acid did not cause any changes in IL-17, COX-2, IL-6, or TNF-\u0026alpha;.\u003c/p\u003e\n\u003cp\u003eThe results presented in Figure 5E-5F demonstrate that the injection of LPS significantly increased the levels of cAMP and PGE2 in the rat hypothalamic homogenate when compared to the NC. However, treatment with Artemisinic acid (IC) did not significantly reduce the levels of cAMP and PGE2 in the rat hypothalamic homogenate. Furthermore, administration of only Artemisinic acid did not result in significant changes in the levels of cAMP and PGE2 in the rat hypothalamic homogenate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eArtemisinic acid could regulate signals of IL-17 pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe findings in Figure 6 demonstrate that the injection of LPS resulted in a significant increase in the expression of \u003cem\u003eIL-17A\u003c/em\u003e, \u003cem\u003eIL-17F\u003c/em\u003e, \u003cem\u003eACT1\u003c/em\u003e, and \u003cem\u003eHsp90a\u003c/em\u003e in the spleen tissue of rats, while the expression of ERK showed a slight reduction with no significant change. However, treatment with Artemisinic acid led to a decrease in the levels of \u003cem\u003eIL-17A\u003c/em\u003e, \u003cem\u003eIL-17F\u003c/em\u003e, \u003cem\u003eACT1\u003c/em\u003e, and \u003cem\u003eHsp90a\u003c/em\u003e in the spleen tissue compared to the IC, and there was a significant increase in the expression of \u003cem\u003eERK\u003c/em\u003e. It is important to note that administration of Artemisinic acid alone did not cause any changes in the expression of \u003cem\u003eIL-17A\u003c/em\u003e,\u003cem\u003e\u0026nbsp;IL-17F\u003c/em\u003e, \u003cem\u003eACT1\u003c/em\u003e, \u003cem\u003eERK\u003c/em\u003e, or \u003cem\u003eHsp90a\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe application of computer-aided drug design methods has been shown to effectively reduce drug development time and screening costs\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e. Among the various methods used in computer-aided drug design, the network pharmacology method is widely recognized \u003csup\u003e13\u003c/sup\u003e. In our research, we utilized the network pharmacology method to simulate the potential of Artemisinic acid as a drug and predict its molecular targets. These predicted targets have the potential to treat LPS-induced inflammation, and it was observed that the targets involved in treating LPS-induced inflammation were enriched in the IL-17 signaling pathway. Our findings suggest that the activity of Artemisinic acid may be linked to the regulation of the IL-17 signaling pathway in the splenic organ (Figure 2).\u003c/p\u003e\n\u003cp\u003eLPS is a reagent commonly used to induce immune stress in the spleen \u003csup\u003e14\u003c/sup\u003e. Injection of LPS in rats has been shown to cause changes in general vital signs, such as fever and decreased spontaneous activities \u003csup\u003e15, 16\u003c/sup\u003e. Body temperature and independent activities are often used as reference indicators to evaluate LPS-induced inflammation. In our model, we observed a significant increase in body temperature and a noticeable decrease in the frequency of independent activities after injecting rats with LPS, indicating successful mimicry of inflammation. Treatment with Artemisinic acid effectively inhibited the rise in body temperature and increased the frequency of independent activities in rats (Figure 3A and 3B). These findings suggest that Artemisinic acid may have the potential to counteract the adverse changes induced by LPS in body temperature and independent activities.\u003c/p\u003e\n\u003cp\u003eEarly detection and diagnosis of inflammation is crucial \u003csup\u003e17\u003c/sup\u003e. Hematological analysis shows an increase in white blood cell (WBC) count, as well as an increase in the NEUT# and NEUT% in the white blood cell classification, which aids in early detection and diagnosis \u003csup\u003e18, 19\u003c/sup\u003e. In this study, it was observed that Artemisinic acid effectively reduced blood WBC, NEUT#, and NEUT% (Figure 4), suggesting that Artemisinic acid may have potential in inhibiting the elevation of WBC, NEUT#, and NEUT% induced by LPS.\u003c/p\u003e\n\u003cp\u003eIL-17, COX-2, IL-6, TNF-\u0026alpha;, cAMP, and PGE2 are closely associated with the inflammation induced by LPS, exhibiting an overexpression trend in this experiment \u003csup\u003e20, 21\u003c/sup\u003e. Inflammatory factors play a crucial role in the inflammatory response, with IL-17, COX-2, IL-6, and TNF-\u0026alpha; being secreted by immune organs \u003csup\u003e2\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e. When the immune system is under stress, the up-regulation of IL-17 and IL-6 in the blood is linked to the activation of immune cells in the spleen, leading to systemic inflammation \u003csup\u003e23, 24\u003c/sup\u003e. The overexpression of IL-17 and IL-6 triggers an increase in the expression of COX-2 and promotes the expression of PGE2 in the body \u003csup\u003e25\u003c/sup\u003e. In our experiments, we observed an overexpression of IL-17, COX-2, IL-6, cAMP, and PGE2. Treatment with artemisinic acid significantly reduced the levels of IL-17 and COX-2 (Figure 5). Notably, IL-17 serves as the initiating factor of the IL-17 signaling pathway, while COX-2 acts as the downstream factor of this pathway (https://www.kegg.jp/). These findings provide valuable insights into the regulatory role of artemisinic acid in the IL-17 signaling pathway.\u003c/p\u003e\n\u003cp\u003eThe IL-17 family is a significant cytokine family in humans, consisting of six related cytokines: IL-17A to IL-17F\u0026nbsp;\u003csup\u003e26\u003c/sup\u003e. These cytokines have diverse and complex functions. While many IL-17 family members contribute to inflammation and play a role in defending against pathogens and immune-mediated diseases, they also play a part in maintaining mucosal integrity, responding to allergens, reducing inflammation, and regulating lymphocyte function. They can act in an autocrine manner and influence the function of other cytokines, including other IL-17 family members. LPS, a bacterial product, is a crucial pathogenic factor. The IL17 gene is primarily produced by Th17 cells but can also be expressed by neutrophils. It exerts an immunoprotective effect against fungi and bacteria and promotes the body\u0026apos;s immune response \u003csup\u003e27, 28\u003c/sup\u003e. Therefore, overexpression of IL-17 and activation of the IL-17 signaling pathway serve as protective mechanisms against LPS-induced damage to the body.\u003c/p\u003e\n\u003cp\u003eThe functions of IL-17A and IL-17F are well understood. IL-17A and IL-17F have the ability to form homo- and heterodimers, which bind to the IL-17 receptor and activate downstream signaling pathways. This activation leads to the release of pro-inflammatory cytokines \u003csup\u003e29\u003c/sup\u003e. The recruitment of IL-17A-IL-17F stimulates immune cell recruitment of ACT1-Hsp90a, resulting in an increase in the release of cellular inflammatory factors \u003csup\u003e30\u003c/sup\u003e and promoting the secretion of COX-2 and IL-6. When the body is infected and the body temperature rises significantly, ACT1 and Hsp90a levels also rise significantly \u003csup\u003e5\u003c/sup\u003e. However, in the IL-17 signaling pathway, ERK inhibits the secretion of cytokines COX-2 and IL-6. Therefore, ERK may exhibit insignificant changes or low expression \u003csup\u003e31\u003c/sup\u003e. In rats injected with LPS, the expression of IL-17A, IL-17F, ACT1, and Hsp90a in the spleen tissue visibly increased. After treatment with Artemisinic acid, the expression of IL-17A, IL-17F, ACT1, and Hsp90a markedly reduced, while the expression of ERK increased (Figure 6). This suggests that the activity of Artemisinic Acid may be related to the regulation of IL-17A, IL-17F, ACT1, Hsp90a, and ERK in the IL-17 signaling pathway.\u003c/p\u003e\n\u003cp\u003eThe effects of administering Artemisinic acid were studied only on rats. No effects were found on body temperature, voluntary activity, hematological WBC counts, NEUT# and NEUT% in white blood cell classification, IL-17, COX-2, IL-6, TNF-\u0026alpha;, cAMP, PGE2, ACT1, Hsp90a, or ERK (Figure 2-6). These results indicate that Artemisinic acid is not toxic, and all the significant changes observed in the experiment can be attributed to the pharmacological activity of Artemisinic acid.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study utilized network pharmacology to simulate the activity of Artemisinic acid, suggesting a potential connection between its mechanism and the regulation of the IL-17 signaling pathway. Subsequent experimental verification confirmed that Artemisinic acid effectively improved immune stress symptoms in rats, reduced inflammation, and regulated IL-17, ACT1, Hsp90a, ERK, and COX-2 signals, thereby modulating the IL-17 signaling pathway. Importantly, preliminary results indicated the safety of Artemisinic acid application. In conclusion, these findings demonstrate that Artemisinic acid is a safe compound with immunomodulatory activity, potentially achieved through the regulation of the IL-17 signaling pathway.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\" style=\"margin-right: calc(26%); width: 74%;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eLPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eLipopolysaccharide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eIL-17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eInterleukin-17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eWBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eWhite blood cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNEUT#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eNumber\u0026nbsp;of\u0026nbsp;neutrophils\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eNEUT%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eProportion of\u0026nbsp;neutrophils\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eCOX-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eCyclooxygenase-2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eIL-6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eInterleukin-6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eTumor necrosis factor-\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003ecAMP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eCyclic adenosine monophosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003ePGE2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003eProstaglandin E-2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cem\u003eACT1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003e\u003cem\u003eNF-kB Activator 1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cem\u003eHsp90a\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003e\u003cem\u003eHeat shock protein HSP 90-alpha\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cem\u003eERK\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 516px;\"\u003e\n \u003cp\u003e\u003cem\u003eExtracellular regulated protein kinases\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eData Availability\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data that support the findings of this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConflicts of Interest\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding Statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Chongqing basic scientific research business fee (jbky20200014)\u0026nbsp;,Chongqing General Research and Development Project (cstc2019jxjl-jbky10005)\u0026nbsp;and\u0026nbsp;Chongqing Municipal Health Commission Chongqing Key Discipline Construction Fund of Traditional Chinese Medicine(Pharmacology and Toxicology of Traditional Chinese Medicine).\u003c/p\u003e\n\u003cp\u003eACKNOWLEDGMENTS\u003c/p\u003e\n\u003cp\u003eWe thank RuxiaTu and Chonggang Huang, members of the Institute of pharmacology and toxicology of Traditional Chinese Medicine, Chongqing Academy of Chinese Materia Medica.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eX Zhang, Y Zhao, L Guo, Z Qiu, L Huang, X Qu et al. 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Interleukin-17 antagonist attenuates lung inflammation through inhibition of the ERK1/2 and NF-\u0026kappa;B pathway in LPS-induced acute lung injury. \u003cem\u003eMolecular Medicine Reports\u003c/em\u003e. 16:2225-2232; 10.3892/mmr.2017.6837 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3926665/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3926665/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eArtemisinic acid is enriched in the well-known herbaceous plants \u003cem\u003eArtemisia annua \u003c/em\u003eL. Due to the negtive anti-malarial activity of Artemisinic acid , it is only used as raw material for the synthesis of Artemisinin in industry. In fact, Artemisinic acid showed excellent effects on immune inflammation, but its mechanism is still unknown. In order to further develop and utilize Artemisinic acid, the reseach on its mechanism is conducted. In this study, network pharmacology methods were performed to analyze the potential targets of Artemisinic acid and the signaling pathways that regulate inflammation. The classic inflammation experimental methods were used, for example, Lipopolysaccharide (LPS) induces inflammation in rats, and the changes in general vital signs, hematology, cytokines, and key molecules of Interleukin-17(IL-17) signaling pathway were observed after Artemisinic acid remedy. In addition, the toxic effects of Artemisinic acid was alsoinvestigated.Research has demonstrated that artemisinin is a secure compound capable of inhibiting inflammation triggered by LPS. The inhibition of inflammation mechanism may be associated with the regulation of IL-17, \u003cem\u003eACT1\u003c/em\u003e, \u003cem\u003eHsp90a\u003c/em\u003e, \u003cem\u003eERK\u003c/em\u003e, and COX-2 signaling within the IL-17 signaling pathway.\u003c/p\u003e","manuscriptTitle":"Mechanism of Artemisinic acid in regulating inflammation: Insights from network pharmacology and experimental validation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-03 08:29:42","doi":"10.21203/rs.3.rs-3926665/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"85e70c10-75a8-403f-8aee-0af9619d318f","owner":[],"postedDate":"March 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28728572,"name":"Biological sciences/Drug discovery"},{"id":28728573,"name":"Biological sciences/Immunology"},{"id":28728574,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-03-03T08:29:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-03 08:29:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3926665","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3926665","identity":"rs-3926665","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}