| Allergic rhinitis(AR)is a chronic non-infectious inflammatory disease of the nasal mucosa that occurs in atopic individuals after exposure to allergens and is mainly mediated by allergen-specific Ig E and involves multiple immune cells and cytokines in the body.The main clinical manifestations of AR include paroxysmal sneezing,runny nose,nasal itching and nasal congestion,may be accompanied by various complications such as asthma,chronic sinusitis and allergic conjunctivitis,causing great troubles to people’s life and health.Epidemiological surveys showed that about 10-40% people disturbed by AR in the world,not only have poor quality of life,but also cause an increasing socio-economic burden,which has become a global health problem.According to the guidelines for the diagnosis and treatment of AR in China published in 2022,the main clinical drugs used to treat AR including glucocorticoids,antihistamines,antileukotrienes,mast cell membrane stabilizers,decongestants and anticholinergics,or a combination of these drugs.Although the current commonly used clinical drugs can improve patients’ short-term or phasic symptoms to some extent,the above treatment methods still have their own limitations and shortcomings,and long-term uses may lead to drug resistance or other side effects.Therefore,it remains a common goal for clinical practitioners to search safer and more effective treatment options.Ginsenoside Rg3,as the main active component of red ginseng,had been shown various pharmacological effects,including anti-inflammatory,antioxidant,anti-tumor,and neuroprotective,and had also been reported in various immune-related diseases.Among them,several studies had shown that ginsenoside Rg3 had ameliorative effects on allergic airway inflammatory diseases such as asthma.However,the role and mechanism of ginsenoside Rg3 in allergic rhinitis have not been reported yet.Based on the above backgrounds,this study investigated the effect of ginsenoside Rg3 on AR in mice firstly,and then explored the mechanism of ginsenoside Rg3 in protecting AR mice through integrative analysis of transcriptomics and metabolomics,and finally applied molecular biology related techniques through the establishment of in vitro cellular and in vivo animal models to discuss the protective effect and mechanism of ginsenoside Rg3 on AR,which provides a theoretical basis and data support for the further search of safer and more effective AR therapeutic drugs.1.The effect of ginsenoside Rg3 on OVA-induced AR miceThe protective effect of ginsenoside Rg3 on AR mice was first evaluated by using the nasal exposure inhalation tower nebulization method,which fully exposed the nasal cavity of mice and inhaled only a uniform and equal amount of nebulized solution through the nose to establish an OVA-induced AR mice model,and different doses of ginsenoside Rg3 were given by intragastric administration for 7 consecutive days.The results showed that compared with the model group(OVA group),the low ginsenoside Rg3 dose group could reduce the number of sneezing and eosinophils in the nasal lavage fluid and decrease the level of IL-13 in the serum of AR mice to some extent;the number of sneezing and scratching noses and the number of inflammatory cells in the nasal lavage fluid of AR mice were significantly reduced in the medium and high ginsenoside Rg3 dose groups.In the high ginsenoside Rg3 dose group,the inflammatory infiltration of the nasal mucosa was significantly improved,and the levels of IL-4 and IL-13 in the serum were significantly reduced;in addition,the levels of total Ig E,IL-5 and MDA in the serum were significantly reduced,and the levels of SOD were significantly increased.It could be inferred that ginsenoside Rg3 has a good protective effect on AR mice,and it is dose-dependent in a certain range.2.The mechanism of ginsenoside Rg3 on OVA-induced AR mice based on integrative analysis of transcriptomics and metabolomicsBased on integrative analysis techniques of transcriptomics and metabolomics,canonical correlation analysis(CCA)and Pearson’s correlation coefficient were used as the joint analysis method to analyze the nasal mucosal tissues of normal control group,OVA group and high ginsenoside Rg3 dose group mice,respectively,and to explore the effect of ginsenoside Rg3 on the expression levels of m RNA and endogenous metabolites in AR mice,as well as the interactions of related pathways.The results showed that the expression of m RNA and endogenous metabolites in the nasal mucosa of AR mice was significantly altered compared with normal control mice.Among these,the expression levels of 3 genes including COL1A1,COL1A2 and COL3A1 and 4 metabolites including glycerophosphocholine,PC(16:0/16:0),lyso PC(18:1(9Z)/0:0)and PE(P-18:0/20:4(5Z,8Z,11 Z,14Z))were increased and down-regulated after ginsenoside Rg3 intervention significantly,while the expression levels of 9 genes including CSN3,CYP26B1,DNASE1,HMOX1,KLF15,LCN2,PIM3,PIP and S100A8,and other 4 metabolites including glutathione,stearidonic acid,α-linolenic acid and β-retinol were reduced and up-regulated by the intervention of ginsenoside Rg3 significantly.It could be inferred that the above 12 differential genes and 8 differential metabolites were the key targets of the protective effect of ginsenoside Rg3 on AR mice.Further analysis enriched 4 pathways,including retinol metabolism,glycerophospholipid metabolism,ECM-receptor interaction andα-linolenic acid metabolism,and gene-metabolite interaction networks,providing a theoretical basis for investigating the protective effects of ginsenoside Rg3 on AR mice.After literature review,it was found that most of the targets obtained from the integrative analysis interacted with antioxidant-related proteases including Nrf2 and HO-1,and it was hypothesized that ginsenoside Rg3 might have a protective effect on AR mice by modulating the Nrf2/HO-1 pathway to improve the level of oxidative stress.3.The protective effect of ginsenoside Rg3 on AR by regulating Nrf2/HO-1pathwayThe H2O2-induced oxidative stress model of human nasal epithelial cells(HNEp C)and the OVA-induced AR mice model were established respectively,and ML385 was applied to specifically inhibit the expression of key protein Nrf2 to verify the protective effect of ginsenoside Rg3 on AR by regulating the Nrf2/HO-1 pathway.In cellular experiments,the results of ELISA and western blot showed that ginsenoside Rg3 could significantly increase the relative expression levels of protein of Nrf2 and HO-1 with antioxidant effects in HNEp C,while ML385 could significantly inhibit its regulatory effects on Nrf2 and HO-1,suggesting that ginsenoside Rg3 might have positive effect on anti-oxidative stress by regulating Nrf2 and HO-1 which was the downstream of Nrf2.In animal experiments,the results of nasal symptoms,histopathological examination,ELISA,immunohistochemistry and western blot showed that ginsenoside Rg3 could promote the relative expression of protein of Nrf2 and HO-1 in the nasal mucosa of AR mice,while ML385 significantly inhibited its regulatory effect on Nrf2 and its downstream HO-1,which was similar to the trend of in cellular experiments.It could be inferred that ginsenoside Rg3 might mainly ameliorate oxidative stress through regulating the Nrf2/HO-1 pathway and thus provide a protective effect on AR mice.In summary,this study evaluated the protective effect of ginsenoside Rg3 on OVA-induced AR mice,and explored its mechanism from multiple perspectives,including genes,metabolites and signaling pathways,using integrative analysis of transcriptomics and metabolomics.On this basis,the regulatory effect of ginsenoside Rg3 on Nrf2/HO-1 signaling pathway were verified by HNEp C oxidative stress model and AR mice model.The medicinal application of ginsenoside Rg3 was expanded through this study,and it also provided a natural,effective,multi-pathway,multi-target innovative drug candidate for the treatment of AR. 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