Ginsenoside Rb1 alleviates airway inflammation in asthma by regulating mitochondrial dysfunction through SIRT1/PGC-1α and PI3K/AKT signaling pathway

Ginsenoside Rb1 alleviates airway inflammation in asthma by regulating mitochondrial dysfunction through SIRT1/PGC-1α and PI3K/AKT signaling pathway | 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 Ginsenoside Rb1 alleviates airway inflammation in asthma by regulating mitochondrial dysfunction through SIRT1/PGC-1α and PI3K/AKT signaling pathway Huiwen Li, Ying Piao, Qiaoyun Bai, Xue Han, Lin Shen, Xiaohan Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3957667/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 Aim of this study is to investigate whether Ginsenoside Rb1 attenuates cockroach extract (CRE) induced asthma by interfering with mitochondrial dysfunction. After induction of CRE, mice were administrated different dose of Rb1. HE staining, ELISA and flow cytometry analysis showed that, the inflammatory cell infiltration, total IgE and CRE specific IgE in serum, and inflammatory cytokines in bronchoalveolar lavage fluid (BALF) were effectively inhibited by Rb1. Through Western blot, TUNEL and immunofluorescence co-localization assay, we observed Rb1 also inhibited endogenous reactive oxygen species (ROS), tightly associated with increased superoxide dismutase (SOD), catalase (CAT) levels, and decreased malondialdehyde (MDA). Subsequently, the silent information regulator Sirtuni1(SIRT1)/peroxisome proliferator-activated receptor-γ coactivator α (PGC-1α) pathway were activated, whereas, phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were alleviated. All of which led to mitochondria dysfunction via promoting mitochondrial fusion protein Mitofusion 1 (MFN1) and inhibiting dynamin-related protein 1 (DRP1) expression and apoptosis in lungs. In BEAS-2B cells, Rb1 played a similar role as SIRT1 agonist (SRT1720), including mitochondrial membrane potential enhancement, mitochondrial ROS and DRP1 translocation to mitochondria decrease. Our findings suggest that Rb1 maintains mitochondria integrity by activating SIRT1/PGC-1α, inhibiting PI3K/AKT, thereby ameliorates asthmatic airway inflammation. Ginsenoside Rb1 SIRT1 PGC-1α PI3K/AKT asthma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Bronchial Asthma is characterized by chronic airway epithelial inflammation that seriously endangers human health [ 1 ] . Cockroaches are insects that are often seen in modern urban environments and are also a common class of allergens that can cause allergic asthma [ 2 ][ 3 ] . ROS are mainly generated from mitochondria, and excessive of them will mediate mitochondrial dysfunction, including mitochondrial fission/fusion imbalance, decreased mitochondrial membrane potential and apoptosis, which are important factors leading to airway epithelial injury in asthma [ 4 ] . Therefore, it is of great clinical significance to investigate a new therapeutic agents targeting CRE-induced mitochondrial dysfunction in asthmatic airway inflammation. Sirtunis family proteins belong to class III histone deacetylases [ 5 ] , that plays an anti-inflammatory role in asthma [ 6 , 7 , 8 ] . Utilizing SIRT1 agonists in ovalbumin (OVA)-induced asthmatic mice both reduced peri-airway inflammatory cell infiltration and inflammatory cytokine production [ 6 ] . NAD + can attenuate oxidative stress-mediated neuroinflammatory vascular dementia by activating the SIRT1/PGC-1α pathways to inhibit ROS production by microglia after chronic brain hypoperfusion [ 9 ] . PGC-1α is a transcriptional co-activator that can be activated by the upstream protein SIRT1, whose functions include oxidative phosphorylation and catalytic elimination of ROS, and whose mechanism of action has been widely studied in mitochondrial research [ 10 ] . Strom [ 11 ] et al. reported that PGC-1α can activate its mediation of Nfe212 by interacting with expression of the antioxidant enzyme SOD2, scavenging excess mtROS and effectively preventing mitochondrial oxidative stress. The important role of the SIRT1/PGC-1α signaling axis in anti-inflammatory and anti-oxidative stress can be seen. However, the mechanism of action of the SIRT1/PGC-1α axis in asthma requires further exploration. Ginsenoside Rb1 belongs to the protopanaxadiol (PPD) class of compounds and is derived from the rhizome of the perennial herb Ginseng in the Wujia family, with a variety of pharmacological activities such as anti-inflammatory and anti-apoptotic [ 12 , 13 ] . In OVA-induced asthma, Rb1 is able to suppress the inflammatory changes and hyperresponsiveness in airways [ 14 ] . In recent years, studies of Rb1 regulation of mitochondrial dysfunction have been widely reported [ 15 , 16 ] . Proteomic analysis revealed that Rb1 were able to inhibit ROS generation by suppressing mitochondrial complex I in the mouse model of heart disease [ 16 ] . Rajput et al. [ 15 ] suggested that Rb1 enhances the oxidative defense system by reducing ROS, MDA and hydrogen peroxide (H 2 O 2 ) levels; while downregulating Bcl-2 related X protein (Bax), mitochondria-associated apoptotic protein Caspase-3 and Caspase-9 expression, and reversing deoxynivalenol (DON)-induced oxidative stress and excessive apoptosis in splenocytes. In addition, induction of mouse embryonic fibroblasts with Rb1 resulted in a dose-dependent significant increase in both SIRT1 and PGC-1α [ 17 ] . Rb1 also effectively inhibited the expression of the PI3K/AKT proteins and phosphorylation of NF-κB, thereby suppressing acetaminophen (APAP)-induced inflammation in acute liver injury response, which acts as a hepatoprotective agent [ 18 ] . However, the mechanism of action of Rb1 in suppressing mitochondrial fission, fusion and apoptosis still ned to be investigated. Aim of our research was to verify that Rb1 ameliorates mitochondrial dysfunction through the SIRT1/PGC-1α and PI3K/AKT pathways thereby inhibiting asthmatic airway inflammation. This will facilitate further research on asthma prevention and treatment efforts to follow. Materials and methods 45BALB/c female mice (free of specific pathogens, 6–8 weeks, weighing 20-22g) were supplied from animal feeding center of Yanbian University. Breeding condition was 50–60% relative humidity, room temperature at 22 ± 2°C, 12h alternating day and night. All animal experimental procedures were applied in accordance with the Regulations for the Administration of Laboratory Animals and approved by the Ethics Committee of the College of Medicine of Yanbian University (approval number SYXK (JI) 2020-0009). All methods are reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). Animal model and grouping Mice were randomly divided into 5 groups (n = 9), control group, cockroach extract (CRE) group, ginsenoside Rb1 treated (low/high dose) group, and dexamethasone group. The mice in the CRE group were each treated with CRE (50 µg, Greerlabs, XP46D3A4, German) dissolved in 50 µL of saline on day 1 to day 5 via intranasal (i.n.) administration for sensitization. 50 µL of saline was applied for control group mice. On day 11 to day 15, mice were continuously stimulated with equivalent volume and concentration of CRE (i.n.) daily under light anesthesia [ 19 ] . Mice in the Rb1-treated group were gavaged with Rb1 (10 and 20 mg/kg, B21050, yuanye Bio-Technology Co. Shanghai, China) for 5 d, 1 time/d [ 14 ] . The control group used 50 µL saline. Mice were sacrificed by cervical dislocation under deep anesthesia using halothane 2 hours after the last challenge for further analysis. Sample collection BALF was obtained from 3 randomly selected mice in each group, then were stained with Diff-quick (G1541, Solarbio, Beijing, China) to calculate the proportion of eosinophils. Other 3 mice were extracted for serum analysis form peripheral blood, mediastinal lymph nodes (mLNs) and right lung for molecular biology analysis. The left lungs were applied by histochemical staining. The rest of lungs were frozen for further analysis. ELISA The cytokine levels from BALF supernatant were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA), including IL-4, IL-5, IL-13 and IFN-γ (M4000B, M5000, M1300CB, MIF00). All with a sensitivity of 2.0pg/mL. Serum CRE-specific and total IgE (SEKR-0019, Solarbio) were also measured [ 20 ] . MDA, CAT, SOD (A003-1-2, A007-1-1, A001-3-2; Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) ELISA assay was performed. Histological analysis Paraffin embedded lung sections were stained with Hematoxylin and Eosin Kit (G1120, Solarbio) to observe airway inflammation. Modified Masson's Trichrome Stainning (G1346, Solarbio) was performed to observe collagen deposition. MTT assay The BEAS-2B cells viability after treatment was applied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) solution as previously described [ 21 ] . Cell culture and treatment The BEAS-2B cells were purchased from the Cell Resource Centre, Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). The cell culture medium was DMEM (vivacell) dissolved with 10% fetal bovine serum (vivacell, Shanghai, China), streptomycin (100 g/mL) and penicillin (100 U/mL), incubated at 37°C and 5% CO 2 . For stimulation, 1 × 10 5 of cells were administered with 60µM of Rb1 for 1h [ 22 ] , or treated with MitoTempo (10µM, HY-112879, MCE, USA) for 30 min [ 23 ] , or with SIRT1 agonist SRT1720 (HY-15145, 1µM, MCE) for 6 h [ 24 ] , and then exposed to 50µg/ml of CRE for 24h [ 19 ] . ROS, mitochondrial morphology, mitochondrial membrane potential assay (MMP), TUNEL Total cellular ROS was detected by incubation with DCFH-DA (10 µM, S0033S, Beyotime) fluorescent probe at 37°C, 10 min. Tissue ROS was detected by Dihydroethidium (DHE) (10 µM,S0063,Beyotime) at 37°C, 5 min. For mitochondrial morphology, intact tubular networks of mitochondria were characterized as not injury, on the contrary, ruptured and spherical morphology of mitochondria defined as fission mitochondria. Mitochondrial length and the DRP1 density were all analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). Cellular mitochondrial ROS (mtROS) was detected by MitoSOX (dye, 5µM, M36008, Thermo Fisher Scientific, Massachusetts, USA) for 10min. For MMP detection, cells were stained with JC-1 (5µM, C2006, Beyotime) at 37°C for 15min and calculate the red/green fluorescence intensity ratio. Apoptosis was detected using the Dead End fluorescent TUNEL kit (C1089, Beyotime). All images were photographed using the Cytation5 Cell Imaging Microplate Detection System (BioTek, Inc., Winooski, VT, USA). Western blotting After extraction, proteins (20 µg) was applied by 12% SDS-PAGE. The gel was incubated with antibodies: SIRT1 (110304, Abcam, USA), Cleaved Caspase-3 (32042, Abcam), PGC-1α (77210, Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 5174, CST, USA), Bax (182734, Abcam), DRP1 (ab184247, Abcam), Bcl-2 (182858, Abcam), MFN1 (221661, Abcam) and p-DRP1 (Ser616) (3455, CST, USA), PI3K(4292, CST), p-PI3K(17366, CST), AKT (8805, Abcam) and p-AKT(38449, Abcam) for 4℃ overnight. Then incubated with antibodies HRP-goat anti-rabbit antibody (5151, CST, USA) or HRP-anti-mouse antibody (5257, CST). Grey-scale values were measured using Quantity One software (BioRad, Hercules, CA, USA). Flow cytometry BAL cells and single cells suspension of mLNs were incubated by surface marker FITC-CD4 antibody (11-0041-82, Invitrogen, USA). Cells were then treated with Intracellular Fixation and Permeabilisation Kit (88-8824-00; Invitrogen), followed by staining by APC-IL-4 antibody (554436, BD, USA) and PE-CY7-IFN-γ antibody (25-7311-82, Invitrogen, USA). Samples were finally collected using a CytoFLEX flow cytometer (Beckman Coulter, Inc., CA, USA) and calculated for the ratios of IL-4 + and IFN-γ + in the CD4 + cells using Cytoexpert 2.4 software. Immunofluorescence assay For mitochondrial imaging, treated cells were incubated with the mitochondrial marker MitoTracker Red (M7512, Thermo) at 37°C, 30 min. For co-staining of MFN1 and mitochondria, lung sections firstly incubated with MitoTracker Red, washed and incubated at 4°C (overnight) with rabbit anti-MFN1 antibody (57602, Abcam). For the DRP1 translocation assay, cells were stained with MitoTracker Red and then incubated with rabbit anti-DRP1 antibody (8570S, CST). At last, tissue sections were treated by DAPI (P0131, Beyotime). The slides were photographed by Cytation5. Fluorescence density was analyzed by Image J. Statistical analysis Data are applied as the mean ± standard deviation of three independent experiments. Significance of differences between the two groups was determined by Student t-test with SPSS 19.0 software (IBM Co., Armonk, NY, USA). Multiple comparisons were performed using ANOVA or Wilcoxon rank sum test. We considered statistically significant when the p -value was < 0.05. Results Rb1 attenuates airway inflammation CRE-induced in asthma To investigate the modulatory effect of ginsenoside Rb1 on airway inflammation in CRE-induced asthmatic mice, we observed histopathological changes around the airway and Th1/Th2 balance in BALF and mediastinal lymph nodes (mLNs). A CRE-induced asthmatic inflammation mouse model was established (Fig. 1 A). Rb1 treatment inhibited eosinophil occupancy (Fig. 1 B-C) and Th2 (IL-4, IL-5, IL-13) cytokines in BALF and promoted the Th1 (IFN-γ) cytokine compared to the CRE model group (Fig. 1 D). Rb1 also reduced serum total IgE and CRE-specific IgE (Fig. 1 E), and reduced the ratios of IL-4 + cells and increased the ratios of IFN-γ + cells in CD4 + cells in mLNs (Fig. 2 A-B). Pathological staining illustrated that Rb1 reduced infiltration of inflammatory cells and deposition of collagen (Fig. 2 C). Taken together, Rb1 inhibited airway inflammation, down-regulated serum IgE levels, reversed Th1/Th2 imbalance and alleviated CRE-induced airway inflammation in asthma. Rb1 attenuates oxidative stress To define the effects of Rb1 under CRE induction, we measured the levels of oxidative stress-related agents and the antioxidant enzymes in lung tissues. Resultantly, Rb1 inhibited ROS and MDA production compared to the CRE model group (Fig. 3 A-B) and significantly upregulated both SOD and CAT levels in tissues (Fig. 3 C-D). Therefore, Rb1 reduced oxidative damage attributed to the counteraction of ROS; and at the same time, upregulation of antioxidant enzymes. Rb1 inhibits mitochondrial fission by activating the SIRT1/PGC-1α and decreasing PI3K/AKT axis We evaluated modulatory role of Rb1 on SIRT1/PGC-1α and PI3K/AKT signaling, followed by downstream mitochondrial dynamics by immunoblotting analysis. As delineated in Fig. 4 A-B, Rb1 activated SIRT1/PGC-1α protein expression compared to the CRE group, whereas inhibited p-PI3K, p-AKT protein expression. Moreover, Rb1 inhibited DRP1, P-DRP1 (ser616) expression but promoted MFN1 expression (Fig. 4 C-D). Consistently, immunofluorescence co-staining analysis showed that deficiency of MFN1 on mitochondria caused by CRE-induced asthma was evidently reversed by the treatment of both Rb1 and Dex (Fig. 4 E). Regarding the effect of Rb1 on airway inflammation and oxidative stress, we suggest that Rb1 alleviates asthmatic inflammation may be associated with the suppression of SIRT1/PGC-1α and PI3K/AKT pathways. Rb1 alleviates asthmatic apoptosis in airway epithelial Western blotting indicated that the apoptotic protein levels such as Cleaved Caspase-3 and Bax were significantly reduced in lung tissues after Rb1 treatment, while the level of Bcl-2 increased (Fig. 5 A-B). Similarly, the TUNEL assay of lungs displayed that the most TUNEL-positive cells were on the airway epithelial cells, where oxidative and mitochondrial damage had occurred (Fig. 5 C). Representing the pivotal role of Rb1 on modulating mitochondrial apoptosis in the airway epithelium of asthmatic mice. Rb1 inhibits CRE triggered mitochondrial ROS, mitochondrial membrane potential in BEAS-2B In vitro , we performed BEAS-2B cells subjected to CRE treatment in the presence of Rb1 to simulate in vivo experiments. Figure 6 A MTT assay displayed that Rb1 had no significant toxic effect in cell viability (10, 30, 60µM) intervention compared to the control. The morphology of mitochondria was next observed by the fluorescent dye Mitotracker-Red (Fig. 6 B-C). Rb1 was found to reverse the extent of CRE-stimulated mitochondrial breakage and fragmentation. A similar antioxidative effect of Rb1 was also shown in CRE-induced BEAS-2B cells, which can decrease total ROS and mtROS (Fig. 6 D-G). Analysis of MMP by JC-1 dye showed that the red/green fluorescence ratio decreased after CRE treatment and was significantly improved by Rb1, signifying the recovery of membrane potential (Fig. 6 H-I). Together, suggested that Rb1 can reduce CRE triggered ROS/mtROS accumulation, thereby enhancing MMP and thus preventing against CRE-induced mitochondrial dysfunction. Rb1 inhibits CRE triggered mitochondrial fission and apoptotic rate in BEAS-2B cells through increasing SIRT1/PGC-1α and decreasing PI3K/AKT expression Consistent with above results, Rb1 promoted the SIRT1/PGC-1α protein expression, inhibited the p-PI3K and p-AKT expression (Fig. 7 A-B), and furthermore modulated the mitochondrial fission/fusion imbalance (Fig. 7 C-D). All of which were similar to the effect of the SIRT1 agonists SRT1720 and superoxide scavenger MitoTEMPO. Immunofluorescence co-localization assay revealed that cells exposed to CRE displayed significant co-localization between DRP1 and mitoTracker Red, whereas, notably reversed by Rb1, SRT1720 and MitoTEMPO treatment (Fig. 7 E-F). Immunoblot analysis showed that Rb1 inhibited Cleaved Caspase-3 and Bax expression while promoting Bcl-2 expression (Fig. 8 A-B). Using TUNEL assay, the proportion of TUNEL + cells was evidently decreased after Rb1 treatment (Fig. 8 C-D). These have a tremendous regulatory effect on mitochondrial homeostasis. Collectively, our data demonstrated that the Rb1 attenuates mitochondrial dysfunction by modulating SIRT1/PGC-1α and PI3K/AKT signaling pathways, thereby suppressing asthmatic inflammation. Discussion Here, we validated molecule mechanisms that Rb1 attenuates CRE-induced mitochondrial fission and apoptosis by regulating the SIRT1/PGC-1α, and PI3K/AKT signaling pathways, thereby alleviating asthmatic airway inflammation. Rb1 prevents CRE-induced inflammatory cell infiltration around airway, decreases ROS generation in lung tissues, up-regulates SIRT1/PGC-1α expression, and down-regulates PI3K/AKT expression. Rb1 further reduced DRP1, BAX and Cleaved Caspase-3 expression, and increased MFN1 and Bcl-2 expression. In vitro experiments, Rb1 effectively increased CRE-induced SOD and CAT levels and decreased MDA levels in BEAS-2B cells, while inhibiting ROS/mtROS production and DRP1 translocation to mitochondria. This implies the preservative effect of Rb1 against CRE-induced asthmatic inflammation. PGC-1α has been shown to have a potential therapeutic effect on bronchial asthma [ 25 ] . When BEAS-2B cells were treated with montelukast, which has anti-inflammatory and antioxidant properties, the expression of PGC-1α was significantly increased [ 25 ] . According to Jun et al [ 26 ] , 6-gingerol reversed the arsenic trioxide (As2O3)-induced inhibition of the AMPK/SIRT1/PGC-1α pathway, thereby inhibiting myocardial oxidative stress, inflammation and apoptosis and attenuating the myocardial toxicity of As2O3. On this basis, we have further explored and validated that activation of SIRT1/PGC-1α is effective in alleviating CRE-induced inflammatory lesions in asthma. Moreover, herein, we found that the SIRT1/PGC-1α axis could additionally mediate the protective effects of altered mitochondrial dynamics and apoptosis. This is supported by a review of the literature representing that PGC-1α became a major agent in preserving dynamic homeostasis of mitochondrial [ 27 ] . PGC-lα directly upregulates MFN1 gene transcription by co-activating estrogen-related receptor α on conserved DNA elements [ 27 ] , and immunoprecipitation analysis revealed that PGC-lα also binds to the DRP1 promoter and downregulates DRP1 expression [ 11 ] . Following stimulation by PM2.5 in BEAS-2B cells, rhodopsin increased mitochondrial membrane potential through activation of the SIRT1/PGC-1α signaling axis and inhibited mitochondrial cytochrome c (Cyt-C) release into the cytoplasm-mediated apoptosis [ 28 ] . It has also been reported that the neuroprotective drug BMS-470539 protects against early brain injury by activating the AMPK/SIRT1/PGC-1α pathway to reduce neuronal ROS, Cleaved Caspase-3, and DRP1 expression [ 29 ] . Thus, SIRT1/PGC-1α is an important target for attenuating dysfunction caused by mitochondrial oxidation, fusion fission imbalance and apoptosis. Our study demonstrated that Rb1 could activate SIRT1/PGC-1α axis to inhibit fission/fusion imbalance of mitochondria caused by oxidative stress and reduce airway inflammation in asthma. It has been identified that Rb1 is closely related to mitochondrial dysfunction. Rb1 significantly increases mitochondrial content in myocardium and enhances aerobic cellular respiration by activating the SIRT1 pathway to promote mitochondrial energy metabolism and ATP synthesis, thereby protecting cardiomyocytes [ 30 ] . Rb1 induces browning of 3T3-L1 and primary white adipocytes, enhances PGC-1α expression in brown adipocytes, and thus increases mitochondrial density [ 31 ] . Although, there is no direct evidence for Rb1 on mitochondrial fission and fusion, ginsenoside Rd inhibits oxygen glucose deprivation/reoxygenation-induced overexpression of DRP1 and activation of NLRP3 inflammatory vesicles in BV-2 cells [ 32 ] , thus, we hypothesize that ginsenoside Rb1 also has a modulatory effect on mitochondrial fission and fusion. In the present study, Rb1 effectively inhibited CRE-induced ROS in lung tissues and mtROS in cells. Combined with the support of several studies [ 15 , 16 , 33 ] , it can be inferred that Rb1 may regulate mitochondrial fission fusion in an oxidative stress-mediated manner. Liu et al. [ 34 ] showed that the elevated ROS/mtROS could trigger PM2.5-induced DRP1 protein overexpression and apoptosis in BEAS-2B cells. It is therefore concluded that Rb1 activates the SIRT1/PGC-1α pathway through down-regulation of CRE-induced ROS/mtROS and inhibits DRP1-mediated mitochondrial damage, ultimately achieving a therapeutic effect on asthma-induced airway inflammation. Many studies have shown that inhibition of the PI3K/AKT pathway is an effective way to alleviate mitochondrial fusion imbalance and suppress asthma airway inflammation. Cigarettes exacerbate asthma airway inflammation by activating the PI3K/AKT signaling axis, increasing DRP1 and decreasing MFN2-mediated mitochondrial dysfunction in ASM cells [ 35 ] . In the present study, we also obtained the similar conclusion that Rb1 treatment alleviated asthmatic inflammation through PI3K/AKT pathway deactivation. It is known that there is also a reciprocal regulatory relationship between SIRT1/PGC-1α and PI3K/AKT expression. When resveratrol was applied to diabetic mice, it increased AMPK/SIRT1/PGC-1α and decreased PI3K-AKT protein expression, thereby improving diabetic nephropathy [ 36 ] . Combining application of minocycline and botulinum toxin promotes SIRT1 expression, inactivates the PI3K/AKT signaling pathway, reduces inflammation and oxidative stress in lipopolysaccharide (LPS)-treated primary microglia, and alleviates neuropathic pain due to spinal cord injury [ 37 ] . The PI3K/AKT pathway and apoptosis were reported to be inhibited following Rb1 treatment in a rabbit model of osteoarthritis [ 38 ] . We therefore hypothesized that upregulation of SIRT1/PGC-1α expression by Rb1 along with inhibition of PI3K/AKT expression could effectively alleviate oxidative stress-induced imbalance in mitochondrial fission and fusion. Conclusion We found that Rb1 could improve asthmatic inflammation via decreased CRE-induced oxidative stress followed by mitochondrial dysfunction and apoptosis. Enhancement of SIRT1/PGC-1α protein expression and suppression of PI3K/AKT expression may be a underlying mechanism. Thus Rb1 has a critical role in ameliorating the development of asthma and may become a promising pharmaceutical preparations for asthma. Declarations Conflicts of interest All authors declare no conflict of interest. Authorship Contributions Participated in research design: H.L., Y.P. and Q.B. Conducted experiments: H.L., Q.B., X.H., L.S. Contributed new reagents or analytic tools: X.L. and L.S. Performed data analysis: H.P. and Y.P. Wrote or contributed to the writing of the manuscript: G.Y., Y.P. and Y.S. Funding This study was funded by National Natural Science Foundation of China (NSFC: 82060004 and 82160004). Jilin Provincial Department of Education Project (JJKH20220547KJ, JJKH20230635KJ and JJKH20240690KJ). Higher Education Discipline Innovation Project (111 Project, No. D18012). Data availability statement Data is provided within the manuscript. 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Y., Lim, J. H., Youn, H. H., Hong, Y. A., Yang, K. S., Park, H. S., Chung, S., Ko, S. H., Shin, S. J., Choi, B. S., Kim, H. W., Kim, Y. S., Lee, J. H., Chang, Y. S., & Park, C. W. (2013). Resveratrol prevents renal lipotoxicity and inhibits mesangial cell glucotoxicity in a manner dependent on the AMPK-SIRT1-PGC1α axis in db/db mice. Diabetologia, 56(1), 204–217. https://doi.org/10.1007/s00125-012-2747-2 Yu, Z., Liu, J., Sun, L., Wang, Y., & Meng, H. (2021). Combination of Botulinum Toxin and minocycline Ameliorates Neuropathic Pain Through Antioxidant Stress and Anti-Inflammation via Promoting SIRT1 Pathway. Frontiers in pharmacology, 11, 602417. https://doi.org/10.3389/fphar.2020.602417 Hossain, M. A., Alam, M. J., Kim, B., Kang, C. W., & Kim, J. H. (2022). Ginsenoside-Rb1 prevents bone cartilage destruction through down-regulation of p-Akt, p-P38, and p-P65 signaling in rabbit. Phytomedicine : international journal of phytotherapy and phytopharmacology, 100, 154039. https://doi.org/10.1016/j.phymed.2022.154039 Additional Declarations No competing interests reported. Supplementary Files WensternBlotrawdata2.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3957667","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":280918212,"identity":"519877ec-dc1e-4acd-b5df-166a2b77a21d","order_by":0,"name":"Huiwen Li","email":"","orcid":"","institution":"Affiliated Hospital of Yanbian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huiwen","middleName":"","lastName":"Li","suffix":""},{"id":280918213,"identity":"c9fdd086-43de-4e09-9c1f-2e3b8900d9f3","order_by":1,"name":"Ying Piao","email":"","orcid":"","institution":"Affiliated Hospital of Yanbian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Piao","suffix":""},{"id":280918214,"identity":"fa3877e8-807b-449b-9bb4-e76a83190123","order_by":2,"name":"Qiaoyun Bai","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiaoyun","middleName":"","lastName":"Bai","suffix":""},{"id":280918215,"identity":"cf04c049-5e58-4559-9d12-885734cd422e","order_by":3,"name":"Xue Han","email":"","orcid":"","institution":"Affiliated Hospital of Yanbian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Han","suffix":""},{"id":280918216,"identity":"f0a50696-93fb-49b3-80d0-97b4de3c12b5","order_by":4,"name":"Lin Shen","email":"","orcid":"","institution":"Yanbian University Medical College","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Shen","suffix":""},{"id":280918217,"identity":"151983c9-6e76-442a-be5f-7762df47e6db","order_by":5,"name":"Xiaohan Liu","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaohan","middleName":"","lastName":"Liu","suffix":""},{"id":280918218,"identity":"96bd95d5-77bd-426a-bd33-e2f3f2b7d0fb","order_by":6,"name":"Hongmei Piao","email":"","orcid":"","institution":"Affiliated Hospital of Yanbian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongmei","middleName":"","lastName":"Piao","suffix":""},{"id":280918219,"identity":"013d5660-f45f-4258-a9bd-a5ab2bd5bd18","order_by":7,"name":"Guanghai Yan","email":"","orcid":"","institution":"Yanbian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guanghai","middleName":"","lastName":"Yan","suffix":""},{"id":280918220,"identity":"1fec3e98-7858-4474-972c-e1728fa0de27","order_by":8,"name":"Yihua Piao","email":"","orcid":"","institution":"Affiliated Hospital of Yanbian University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yihua","middleName":"","lastName":"Piao","suffix":""},{"id":280918221,"identity":"af58301c-88b9-444e-87fc-224c036199f3","order_by":9,"name":"Yilan Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYNCCAhsGAxDNQ7wWgzTStRwmQYt8e+/h1zwG5xO3SyQwPnjbxiBvTtD8M+fSrHkMbifunJHAbDi3jcFwZwMhLRI5ZsYgLRtuJLBJ87YxJBgcIOSwGWAt50Ba2H8TpYXhRo7xYx6DA2BbmInSYnDmjBnjHINk4509D5sl55yTMNxA0GHtPcYf3lTYyW5nTz744U2ZjTxhhzEwsEkBo8OxgYGxAciRIKweCJg//mBgsCdK6SgYBaNgFIxMAAAJ/j+/b8RK7gAAAABJRU5ErkJggg==","orcid":"","institution":"Yanbian University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yilan","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2024-02-15 05:17:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3957667/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3957667/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53010136,"identity":"0a753292-2162-4109-94f5-120983884523","added_by":"auto","created_at":"2024-03-19 15:25:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":438917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of Rb1 on CRE-induced airway inflammation in asthmatic mice.\u003c/strong\u003e(A) Diagrammatic representation of CRE-triggered mouse model of asthma with or without Rb1 (n = 9). (B-C) Quantitative analysis of eosinophils in BAL cells by Diff-Quick staining. Arrow indicates eosinophils. Scale bar = 200μm. (D) Cytokine (IL-4, IL-5, IL-13, IFN-γ) levels in supernatants of mouse BALF; (E) Levels of total IgE and CRE-specific IgE. Data are applied as the mean ±SD. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. CRE group.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/81e398a291090b2a660de4aa.jpg"},{"id":53010138,"identity":"522189f1-83c2-4519-a02c-b78f9281888f","added_by":"auto","created_at":"2024-03-19 15:25:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":842326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of Rb1 on CRE-induced Th cytokines in asthmatic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Flow cytometric detection of IL-4+ and IFN-γ+ in the CD4+ cells in mediastinal lymph nodes (mLNs). (C) HE and Masson staining was applied in lung tissue sections. Arrows represent peri-airway inflammatory cells and collagen deposition, respectively. Scale bar = 200μm. Data are Sapplied as the mean ±SD. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 vs. CRE group.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/fa3ad15d5f2ba4c567b8122c.jpg"},{"id":53011139,"identity":"b57fa505-30ba-4c9e-b6a0-eed7ce18c04c","added_by":"auto","created_at":"2024-03-19 15:33:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":327884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of Rb1 on CRE-induced oxidative stress. \u003c/strong\u003e(A) Detection of ROS production on lung sections by fluoresence assay. Scale bar = 200μm. (B-D) ELISA assay for MDA, CAT and SOD in BALF supernatants. Data are applied as the mean ±SD. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. CRE group.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/408f109e4214fbdb113a31e1.jpg"},{"id":53012466,"identity":"825dbb8e-a80f-496b-aa34-c2e730d5a661","added_by":"auto","created_at":"2024-03-19 15:41:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1150402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of Rb1 on mitochondrial fission by activating the SIRT1/PGC-1α, and PI3K/AKT axis.\u003c/strong\u003e (A) Immunoblotting of SIRT1, PGC-1α, p-PI3K, p-AKT and GAPDH. (B) Relative densities calculated using Quantity One software. (C) Immunoblots of p-DRP1 (ser616), DRP1, MFN1 and GAPDH. (D) Relative densities calculated. (E) Co-localization of MitoTracker Red and MFN1 in the lung tissues. Scale bar = 200μm. Data are applied as the mean ±SD. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. CRE group.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/cf49bdb270c477aefa2b3c20.jpg"},{"id":53010139,"identity":"fc244922-2878-47ab-b018-318d51c99502","added_by":"auto","created_at":"2024-03-19 15:25:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":352458,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of Rb1 on CRE-induced mitochondrial apoptosis.\u003c/strong\u003e(A) Immunoblotting of Bax, Bcl-2, Cleaved Caspase-3 and GAPDH. (B) Calculation of relative densities. (C) TUNEL assay for apoptosis in lung tissues. Scale bar = 200 μm. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. CRE group.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/641bc3238fcf67033aa15ae4.jpg"},{"id":53010142,"identity":"0e04853a-2114-45bc-b7fc-40f4f19c0180","added_by":"auto","created_at":"2024-03-19 15:25:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":990702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of Rb1 on CRE-induced mitochondrial dysfunction in BEAS-2B cells.\u003c/strong\u003e 1×10\u003csup\u003e5\u003c/sup\u003e cells were dealt with different concentrations of Rb1 (10, 30, 60, 100 μm) for 1h, followed by stimulation with CRE (50 μg/ml) for 24h. (A) MTT assay to evaluate the effect of Rb1 on cell viability at different concentrations. (B-C) Mitochondrial morphology using MitoTracker Red staining and mitochondrial length calculation. Scale bar = 25μm. (D-G) Fluorescence assay to detect intracellular total ROS and mtROS (mitochondrial superoxide indicator) production and its quantitative analysis. Scale bar = 200μm. (H-I) MMP detection by JC-1 dye and its quantitative analysis. Scale bar = 200μm. Data are applied as the mean ±SD. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. CRE group.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/1d9f17442d9d05fd104c3518.jpg"},{"id":53010141,"identity":"8f1d11ce-cf21-430f-979f-d8cd4687d734","added_by":"auto","created_at":"2024-03-19 15:25:17","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":886948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of Rb1 on CRE-induced mitochondrial fission in BEAS-2B cells through enhancement in SIRT1/PGC-1α, and PI3K/AKT axis.\u003c/strong\u003e (A) Immunoblotting of PGC-1α, SIRT1, p-AKT, p-PI3K and GAPDH. (B) Relative densities calculated using Quantity One software. (C) Immunoblotting of p-DRP1 (ser616), DRP1, MFN1 and GAPDH (D) Calculated relative densities. (E-F) Co-localization of DRP1 and MitoTracker Red under CRE in BEAS-2B. And the density of mitochondrial puncta calculation. Scale bar = 100μm. Data are applied as the mean ±SD. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. CRE group.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/54d914552d9bd6a12b808e05.jpg"},{"id":53010144,"identity":"5e53fe7c-357d-4f2a-8f63-4df84cb3dbe9","added_by":"auto","created_at":"2024-03-19 15:25:17","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":300563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of Rb1 on CRE-induced mitochondrial apoptosis in BEAS-2B cells.\u003c/strong\u003e (A) Immunoblotting of Bax, Bcl-2, Cleaved Caspase-3 and GAPDH. (B) Calculation of relative densities. (C) TUNEL assay for apoptosis and (D) the proportion of TUNEL-positive cells (%). Scale bar = 200μm. Data are applied as the mean ±SD. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. CRE group.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/f05c9b9c8e0ddd7d4f6e9769.jpg"},{"id":55478259,"identity":"4f68ad5a-1068-4c6a-86d1-81f4f7ab4d76","added_by":"auto","created_at":"2024-04-29 03:22:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1916558,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/7bbc4391-965f-4e58-8861-84f19a1ea91e.pdf"},{"id":53010143,"identity":"1c561393-bb0f-4244-b096-bba727d03fad","added_by":"auto","created_at":"2024-03-19 15:25:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":731509,"visible":true,"origin":"","legend":"","description":"","filename":"WensternBlotrawdata2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3957667/v1/72b1ea751471a2e5e07cde8c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ginsenoside Rb1 alleviates airway inflammation in asthma by regulating mitochondrial dysfunction through SIRT1/PGC-1α and PI3K/AKT signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBronchial Asthma is characterized by chronic airway epithelial inflammation that seriously endangers human health\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Cockroaches are insects that are often seen in modern urban environments and are also a common class of allergens that can cause allergic asthma\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. ROS are mainly generated from mitochondria, and excessive of them will mediate mitochondrial dysfunction, including mitochondrial fission/fusion imbalance, decreased mitochondrial membrane potential and apoptosis, which are important factors leading to airway epithelial injury in asthma\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is of great clinical significance to investigate a new therapeutic agents targeting CRE-induced mitochondrial dysfunction in asthmatic airway inflammation.\u003c/p\u003e \u003cp\u003eSirtunis family proteins belong to class III histone deacetylases\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, that plays an anti-inflammatory role in asthma\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Utilizing SIRT1 agonists in ovalbumin (OVA)-induced asthmatic mice both reduced peri-airway inflammatory cell infiltration and inflammatory cytokine production\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. NAD\u003csup\u003e+\u003c/sup\u003e can attenuate oxidative stress-mediated neuroinflammatory vascular dementia by activating the SIRT1/PGC-1α pathways to inhibit ROS production by microglia after chronic brain hypoperfusion\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. PGC-1α is a transcriptional co-activator that can be activated by the upstream protein SIRT1, whose functions include oxidative phosphorylation and catalytic elimination of ROS, and whose mechanism of action has been widely studied in mitochondrial research\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Strom\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e et al. reported that PGC-1α can activate its mediation of Nfe212 by interacting with expression of the antioxidant enzyme SOD2, scavenging excess mtROS and effectively preventing mitochondrial oxidative stress. The important role of the SIRT1/PGC-1α signaling axis in anti-inflammatory and anti-oxidative stress can be seen. However, the mechanism of action of the SIRT1/PGC-1α axis in asthma requires further exploration.\u003c/p\u003e \u003cp\u003eGinsenoside Rb1 belongs to the protopanaxadiol (PPD) class of compounds and is derived from the rhizome of the perennial herb Ginseng in the Wujia family, with a variety of pharmacological activities such as anti-inflammatory and anti-apoptotic\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In OVA-induced asthma, Rb1 is able to suppress the inflammatory changes and hyperresponsiveness in airways\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In recent years, studies of Rb1 regulation of mitochondrial dysfunction have been widely reported\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Proteomic analysis revealed that Rb1 were able to inhibit ROS generation by suppressing mitochondrial complex I in the mouse model of heart disease\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Rajput et al.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e suggested that Rb1 enhances the oxidative defense system by reducing ROS, MDA and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels; while downregulating Bcl-2 related X protein (Bax), mitochondria-associated apoptotic protein Caspase-3 and Caspase-9 expression, and reversing deoxynivalenol (DON)-induced oxidative stress and excessive apoptosis in splenocytes. In addition, induction of mouse embryonic fibroblasts with Rb1 resulted in a dose-dependent significant increase in both SIRT1 and PGC-1α\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Rb1 also effectively inhibited the expression of the PI3K/AKT proteins and phosphorylation of NF-κB, thereby suppressing acetaminophen (APAP)-induced inflammation in acute liver injury response, which acts as a hepatoprotective agent\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. However, the mechanism of action of Rb1 in suppressing mitochondrial fission, fusion and apoptosis still ned to be investigated.\u003c/p\u003e \u003cp\u003eAim of our research was to verify that Rb1 ameliorates mitochondrial dysfunction through the SIRT1/PGC-1α and PI3K/AKT pathways thereby inhibiting asthmatic airway inflammation. This will facilitate further research on asthma prevention and treatment efforts to follow.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e45BALB/c female mice (free of specific pathogens, 6\u0026ndash;8 weeks, weighing 20-22g) were supplied from animal feeding center of Yanbian University. Breeding condition was 50\u0026ndash;60% relative humidity, room temperature at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 12h alternating day and night. All animal experimental procedures were applied in accordance with the Regulations for the Administration of Laboratory Animals and approved by the Ethics Committee of the College of Medicine of Yanbian University (approval number SYXK (JI) 2020-0009). All methods are reported in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal model and grouping\u003c/h2\u003e \u003cp\u003eMice were randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;9), control group, cockroach extract (CRE) group, ginsenoside Rb1 treated (low/high dose) group, and dexamethasone group. The mice in the CRE group were each treated with CRE (50 \u0026micro;g, Greerlabs, XP46D3A4, German) dissolved in 50 \u0026micro;L of saline on day 1 to day 5 via intranasal (i.n.) administration for sensitization. 50 \u0026micro;L of saline was applied for control group mice. On day 11 to day 15, mice were continuously stimulated with equivalent volume and concentration of CRE (i.n.) daily under light anesthesia\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Mice in the Rb1-treated group were gavaged with Rb1 (10 and 20 mg/kg, B21050, yuanye Bio-Technology Co. Shanghai, China) for 5 d, 1 time/d\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The control group used 50 \u0026micro;L saline. Mice were sacrificed by cervical dislocation under deep anesthesia using halothane 2 hours after the last challenge for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eBALF was obtained from 3 randomly selected mice in each group, then were stained with Diff-quick (G1541, Solarbio, Beijing, China) to calculate the proportion of eosinophils. Other 3 mice were extracted for serum analysis form peripheral blood, mediastinal lymph nodes (mLNs) and right lung for molecular biology analysis. The left lungs were applied by histochemical staining. The rest of lungs were frozen for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe cytokine levels from BALF supernatant were measured using ELISA kits (R\u0026amp;D Systems, Minneapolis, MN, USA), including IL-4, IL-5, IL-13 and IFN-γ (M4000B, M5000, M1300CB, MIF00). All with a sensitivity of 2.0pg/mL. Serum CRE-specific and total IgE (SEKR-0019, Solarbio) were also measured\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. MDA, CAT, SOD (A003-1-2, A007-1-1, A001-3-2; Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) ELISA assay was performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eParaffin embedded lung sections were stained with Hematoxylin and Eosin Kit (G1120, Solarbio) to observe airway inflammation. Modified Masson's Trichrome Stainning (G1346, Solarbio) was performed to observe collagen deposition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eThe BEAS-2B cells viability after treatment was applied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) solution as previously described\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eThe BEAS-2B cells were purchased from the Cell Resource Centre, Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). The cell culture medium was DMEM (vivacell) dissolved with 10% fetal bovine serum (vivacell, Shanghai, China), streptomycin (100 g/mL) and penicillin (100 U/mL), incubated at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. For stimulation, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e of cells were administered with 60\u0026micro;M of Rb1 for 1h\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, or treated with MitoTempo (10\u0026micro;M, HY-112879, MCE, USA) for 30 min\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, or with SIRT1 agonist SRT1720 (HY-15145, 1\u0026micro;M, MCE) for 6 h\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, and then exposed to 50\u0026micro;g/ml of CRE for 24h\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eROS, mitochondrial morphology, mitochondrial membrane potential assay (MMP), TUNEL\u003c/h2\u003e \u003cp\u003eTotal cellular ROS was detected by incubation with DCFH-DA (10 \u0026micro;M, S0033S, Beyotime) fluorescent probe at 37\u0026deg;C, 10 min. Tissue ROS was detected by Dihydroethidium (DHE) (10 \u0026micro;M,S0063,Beyotime) at 37\u0026deg;C, 5 min. For mitochondrial morphology, intact tubular networks of mitochondria were characterized as not injury, on the contrary, ruptured and spherical morphology of mitochondria defined as fission mitochondria. Mitochondrial length and the DRP1 density were all analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). Cellular mitochondrial ROS (mtROS) was detected by MitoSOX (dye, 5\u0026micro;M, M36008, Thermo Fisher Scientific, Massachusetts, USA) for 10min. For MMP detection, cells were stained with JC-1 (5\u0026micro;M, C2006, Beyotime) at 37\u0026deg;C for 15min and calculate the red/green fluorescence intensity ratio. Apoptosis was detected using the Dead End fluorescent TUNEL kit (C1089, Beyotime). All images were photographed using the Cytation5 Cell Imaging Microplate Detection System (BioTek, Inc., Winooski, VT, USA).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eAfter extraction, proteins (20 \u0026micro;g) was applied by 12% SDS-PAGE. The gel was incubated with antibodies: SIRT1 (110304, Abcam, USA), Cleaved Caspase-3 (32042, Abcam), PGC-1α (77210, Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 5174, CST, USA), Bax (182734, Abcam), DRP1 (ab184247, Abcam), Bcl-2 (182858, Abcam), MFN1 (221661, Abcam) and p-DRP1 (Ser616) (3455, CST, USA), PI3K(4292, CST), p-PI3K(17366, CST), AKT (8805, Abcam) and p-AKT(38449, Abcam) for 4℃ overnight. Then incubated with antibodies HRP-goat anti-rabbit antibody (5151, CST, USA) or HRP-anti-mouse antibody (5257, CST). Grey-scale values were measured using Quantity One software (BioRad, Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eBAL cells and single cells suspension of mLNs were incubated by surface marker FITC-CD4 antibody (11-0041-82, Invitrogen, USA). Cells were then treated with Intracellular Fixation and Permeabilisation Kit (88-8824-00; Invitrogen), followed by staining by APC-IL-4 antibody (554436, BD, USA) and PE-CY7-IFN-γ antibody (25-7311-82, Invitrogen, USA). Samples were finally collected using a CytoFLEX flow cytometer (Beckman Coulter, Inc., CA, USA) and calculated for the ratios of IL-4\u003csup\u003e+\u003c/sup\u003e and IFN-γ\u003csup\u003e+\u003c/sup\u003e in the CD4\u003csup\u003e+\u003c/sup\u003e cells using Cytoexpert 2.4 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence assay\u003c/h2\u003e \u003cp\u003eFor mitochondrial imaging, treated cells were incubated with the mitochondrial marker MitoTracker Red (M7512, Thermo) at 37\u0026deg;C, 30 min. For co-staining of MFN1 and mitochondria, lung sections firstly incubated with MitoTracker Red, washed and incubated at 4\u0026deg;C (overnight) with rabbit anti-MFN1 antibody (57602, Abcam). For the DRP1 translocation assay, cells were stained with MitoTracker Red and then incubated with rabbit anti-DRP1 antibody (8570S, CST). At last, tissue sections were treated by DAPI (P0131, Beyotime). The slides were photographed by Cytation5. Fluorescence density was analyzed by Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are applied as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of three independent experiments. Significance of differences between the two groups was determined by Student t-test with SPSS 19.0 software (IBM Co., Armonk, NY, USA). Multiple comparisons were performed using ANOVA or Wilcoxon rank sum test. We considered statistically significant when the \u003cem\u003ep\u003c/em\u003e-value was \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRb1 attenuates airway inflammation CRE-induced in asthma\u003c/h2\u003e \u003cp\u003eTo investigate the modulatory effect of ginsenoside Rb1 on airway inflammation in CRE-induced asthmatic mice, we observed histopathological changes around the airway and Th1/Th2 balance in BALF and mediastinal lymph nodes (mLNs). A CRE-induced asthmatic inflammation mouse model was established (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Rb1 treatment inhibited eosinophil occupancy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C) and Th2 (IL-4, IL-5, IL-13) cytokines in BALF and promoted the Th1 (IFN-γ) cytokine compared to the CRE model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Rb1 also reduced serum total IgE and CRE-specific IgE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and reduced the ratios of IL-4\u003csup\u003e+\u003c/sup\u003e cells and increased the ratios of IFN-γ\u003csup\u003e+\u003c/sup\u003e cells in CD4\u003csup\u003e+\u003c/sup\u003e cells in mLNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Pathological staining illustrated that Rb1 reduced infiltration of inflammatory cells and deposition of collagen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Taken together, Rb1 inhibited airway inflammation, down-regulated serum IgE levels, reversed Th1/Th2 imbalance and alleviated CRE-induced airway inflammation in asthma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRb1 attenuates oxidative stress\u003c/h2\u003e \u003cp\u003eTo define the effects of Rb1 under CRE induction, we measured the levels of oxidative stress-related agents and the antioxidant enzymes in lung tissues. Resultantly, Rb1 inhibited ROS and MDA production compared to the CRE model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B) and significantly upregulated both SOD and CAT levels in tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). Therefore, Rb1 reduced oxidative damage attributed to the counteraction of ROS; and at the same time, upregulation of antioxidant enzymes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRb1 inhibits mitochondrial fission by activating the SIRT1/PGC-1α and decreasing PI3K/AKT axis\u003c/h2\u003e \u003cp\u003eWe evaluated modulatory role of Rb1 on SIRT1/PGC-1α and PI3K/AKT signaling, followed by downstream mitochondrial dynamics by immunoblotting analysis. As delineated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, Rb1 activated SIRT1/PGC-1α protein expression compared to the CRE group, whereas inhibited p-PI3K, p-AKT protein expression. Moreover, Rb1 inhibited DRP1, P-DRP1 (ser616) expression but promoted MFN1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). Consistently, immunofluorescence co-staining analysis showed that deficiency of MFN1 on mitochondria caused by CRE-induced asthma was evidently reversed by the treatment of both Rb1 and Dex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Regarding the effect of Rb1 on airway inflammation and oxidative stress, we suggest that Rb1 alleviates asthmatic inflammation may be associated with the suppression of SIRT1/PGC-1α and PI3K/AKT pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRb1 alleviates asthmatic apoptosis in airway epithelial\u003c/h2\u003e \u003cp\u003eWestern blotting indicated that the apoptotic protein levels such as Cleaved Caspase-3 and Bax were significantly reduced in lung tissues after Rb1 treatment, while the level of Bcl-2 increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). Similarly, the TUNEL assay of lungs displayed that the most TUNEL-positive cells were on the airway epithelial cells, where oxidative and mitochondrial damage had occurred (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Representing the pivotal role of Rb1 on modulating mitochondrial apoptosis in the airway epithelium of asthmatic mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRb1 inhibits CRE triggered mitochondrial ROS, mitochondrial membrane potential in BEAS-2B\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e, we performed BEAS-2B cells subjected to CRE treatment in the presence of Rb1 to simulate \u003cem\u003ein vivo\u003c/em\u003e experiments. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA MTT assay displayed that Rb1 had no significant toxic effect in cell viability (10, 30, 60\u0026micro;M) intervention compared to the control. The morphology of mitochondria was next observed by the fluorescent dye Mitotracker-Red (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). Rb1 was found to reverse the extent of CRE-stimulated mitochondrial breakage and fragmentation. A similar antioxidative effect of Rb1 was also shown in CRE-induced BEAS-2B cells, which can decrease total ROS and mtROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-G). Analysis of MMP by JC-1 dye showed that the red/green fluorescence ratio decreased after CRE treatment and was significantly improved by Rb1, signifying the recovery of membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-I). Together, suggested that Rb1 can reduce CRE triggered ROS/mtROS accumulation, thereby enhancing MMP and thus preventing against CRE-induced mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRb1 inhibits CRE triggered mitochondrial fission and apoptotic rate in BEAS-2B cells through increasing SIRT1/PGC-1α and decreasing PI3K/AKT expression\u003c/b\u003e \u003c/p\u003e \u003cp\u003eConsistent with above results, Rb1 promoted the SIRT1/PGC-1α protein expression, inhibited the p-PI3K and p-AKT expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B), and furthermore modulated the mitochondrial fission/fusion imbalance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D). All of which were similar to the effect of the SIRT1 agonists SRT1720 and superoxide scavenger MitoTEMPO. Immunofluorescence co-localization assay revealed that cells exposed to CRE displayed significant co-localization between DRP1 and mitoTracker Red, whereas, notably reversed by Rb1, SRT1720 and MitoTEMPO treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-F). Immunoblot analysis showed that Rb1 inhibited Cleaved Caspase-3 and Bax expression while promoting Bcl-2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). Using TUNEL assay, the proportion of TUNEL\u003csup\u003e+\u003c/sup\u003e cells was evidently decreased after Rb1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-D). These have a tremendous regulatory effect on mitochondrial homeostasis. Collectively, our data demonstrated that the Rb1 attenuates mitochondrial dysfunction by modulating SIRT1/PGC-1α and PI3K/AKT signaling pathways, thereby suppressing asthmatic inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we validated molecule mechanisms that Rb1 attenuates CRE-induced mitochondrial fission and apoptosis by regulating the SIRT1/PGC-1α, and PI3K/AKT signaling pathways, thereby alleviating asthmatic airway inflammation. Rb1 prevents CRE-induced inflammatory cell infiltration around airway, decreases ROS generation in lung tissues, up-regulates SIRT1/PGC-1α expression, and down-regulates PI3K/AKT expression. Rb1 further reduced DRP1, BAX and Cleaved Caspase-3 expression, and increased MFN1 and Bcl-2 expression. \u003cem\u003eIn vitro\u003c/em\u003e experiments, Rb1 effectively increased CRE-induced SOD and CAT levels and decreased MDA levels in BEAS-2B cells, while inhibiting ROS/mtROS production and DRP1 translocation to mitochondria. This implies the preservative effect of Rb1 against CRE-induced asthmatic inflammation.\u003c/p\u003e \u003cp\u003ePGC-1α has been shown to have a potential therapeutic effect on bronchial asthma\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. When BEAS-2B cells were treated with montelukast, which has anti-inflammatory and antioxidant properties, the expression of PGC-1α was significantly increased\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. According to Jun et al\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, 6-gingerol reversed the arsenic trioxide (As2O3)-induced inhibition of the AMPK/SIRT1/PGC-1α pathway, thereby inhibiting myocardial oxidative stress, inflammation and apoptosis and attenuating the myocardial toxicity of As2O3. On this basis, we have further explored and validated that activation of SIRT1/PGC-1α is effective in alleviating CRE-induced inflammatory lesions in asthma. Moreover, herein, we found that the SIRT1/PGC-1α axis could additionally mediate the protective effects of altered mitochondrial dynamics and apoptosis. This is supported by a review of the literature representing that PGC-1α became a major agent in preserving dynamic homeostasis of mitochondrial\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. PGC-lα directly upregulates MFN1 gene transcription by co-activating estrogen-related receptor α on conserved DNA elements\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, and immunoprecipitation analysis revealed that PGC-lα also binds to the DRP1 promoter and downregulates DRP1 expression\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Following stimulation by PM2.5 in BEAS-2B cells, rhodopsin increased mitochondrial membrane potential through activation of the SIRT1/PGC-1α signaling axis and inhibited mitochondrial cytochrome c (Cyt-C) release into the cytoplasm-mediated apoptosis \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. It has also been reported that the neuroprotective drug BMS-470539 protects against early brain injury by activating the AMPK/SIRT1/PGC-1α pathway to reduce neuronal ROS, Cleaved Caspase-3, and DRP1 expression\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Thus, SIRT1/PGC-1α is an important target for attenuating dysfunction caused by mitochondrial oxidation, fusion fission imbalance and apoptosis.\u003c/p\u003e \u003cp\u003eOur study demonstrated that Rb1 could activate SIRT1/PGC-1α axis to inhibit fission/fusion imbalance of mitochondria caused by oxidative stress and reduce airway inflammation in asthma. It has been identified that Rb1 is closely related to mitochondrial dysfunction. Rb1 significantly increases mitochondrial content in myocardium and enhances aerobic cellular respiration by activating the SIRT1 pathway to promote mitochondrial energy metabolism and ATP synthesis, thereby protecting cardiomyocytes\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Rb1 induces browning of 3T3-L1 and primary white adipocytes, enhances PGC-1α expression in brown adipocytes, and thus increases mitochondrial density\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Although, there is no direct evidence for Rb1 on mitochondrial fission and fusion, ginsenoside Rd inhibits oxygen glucose deprivation/reoxygenation-induced overexpression of DRP1 and activation of NLRP3 inflammatory vesicles in BV-2 cells\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e, thus, we hypothesize that ginsenoside Rb1 also has a modulatory effect on mitochondrial fission and fusion. In the present study, Rb1 effectively inhibited CRE-induced ROS in lung tissues and mtROS in cells. Combined with the support of several studies\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, it can be inferred that Rb1 may regulate mitochondrial fission fusion in an oxidative stress-mediated manner. Liu et al.\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e showed that the elevated ROS/mtROS could trigger PM2.5-induced DRP1 protein overexpression and apoptosis in BEAS-2B cells. It is therefore concluded that Rb1 activates the SIRT1/PGC-1α pathway through down-regulation of CRE-induced ROS/mtROS and inhibits DRP1-mediated mitochondrial damage, ultimately achieving a therapeutic effect on asthma-induced airway inflammation.\u003c/p\u003e \u003cp\u003eMany studies have shown that inhibition of the PI3K/AKT pathway is an effective way to alleviate mitochondrial fusion imbalance and suppress asthma airway inflammation. Cigarettes exacerbate asthma airway inflammation by activating the PI3K/AKT signaling axis, increasing DRP1 and decreasing MFN2-mediated mitochondrial dysfunction in ASM cells\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. In the present study, we also obtained the similar conclusion that Rb1 treatment alleviated asthmatic inflammation through PI3K/AKT pathway deactivation. It is known that there is also a reciprocal regulatory relationship between SIRT1/PGC-1α and PI3K/AKT expression. When resveratrol was applied to diabetic mice, it increased AMPK/SIRT1/PGC-1α and decreased PI3K-AKT protein expression, thereby improving diabetic nephropathy\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Combining application of minocycline and botulinum toxin promotes SIRT1 expression, inactivates the PI3K/AKT signaling pathway, reduces inflammation and oxidative stress in lipopolysaccharide (LPS)-treated primary microglia, and alleviates neuropathic pain due to spinal cord injury\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. The PI3K/AKT pathway and apoptosis were reported to be inhibited following Rb1 treatment in a rabbit model of osteoarthritis\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. We therefore hypothesized that upregulation of SIRT1/PGC-1α expression by Rb1 along with inhibition of PI3K/AKT expression could effectively alleviate oxidative stress-induced imbalance in mitochondrial fission and fusion.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe found that Rb1 could improve asthmatic inflammation via decreased CRE-induced oxidative stress followed by mitochondrial dysfunction and apoptosis. Enhancement of SIRT1/PGC-1α protein expression and suppression of PI3K/AKT expression may be a underlying mechanism. Thus Rb1 has a critical role in ameliorating the development of asthma and may become a promising pharmaceutical preparations for asthma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipated in research design: H.L., Y.P. and Q.B.\u003c/p\u003e\n\u003cp\u003eConducted experiments: H.L., Q.B., X.H., L.S.\u003c/p\u003e\n\u003cp\u003eContributed new reagents or analytic tools: X.L. and L.S.\u003c/p\u003e\n\u003cp\u003ePerformed data analysis: H.P. and Y.P.\u003c/p\u003e\n\u003cp\u003eWrote or contributed to the writing of the manuscript: G.Y.,\u0026nbsp;Y.P. and Y.S.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by National Natural Science Foundation of China (NSFC: 82060004 and 82160004). Jilin Provincial Department of Education Project (JJKH20220547KJ, JJKH20230635KJ and JJKH20240690KJ). Higher Education Discipline Innovation Project (111 Project, No. D18012).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAnimal statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animals were sacrificed with appropriate way and include anesthesia agent used and euthanasia method.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSockrider, M., \u0026amp; Fussner, L. (2020). What Is Asthma?. American journal of respiratory and critical care medicine, 202(9), P25\u0026ndash;P26. https://doi.org/10.1164/rccm.2029P25\u003c/li\u003e\n\u003cli\u003eDo, D. C., Zhao, Y., \u0026amp; Gao, P. (2016). Cockroach allergen exposure and risk of asthma. 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Phytomedicine : international journal of phytotherapy and phytopharmacology, 100, 154039. https://doi.org/10.1016/j.phymed.2022.154039\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Ginsenoside Rb1, SIRT1, PGC-1α, PI3K/AKT, asthma","lastPublishedDoi":"10.21203/rs.3.rs-3957667/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3957667/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAim of this study is to investigate whether Ginsenoside Rb1 attenuates cockroach extract (CRE) induced asthma by interfering with mitochondrial dysfunction. After induction of CRE, mice were administrated different dose of Rb1. HE staining, ELISA and flow cytometry analysis showed that, the inflammatory cell infiltration, total IgE and CRE specific IgE in serum, and inflammatory cytokines in bronchoalveolar lavage fluid (BALF) were effectively inhibited by Rb1. Through Western blot, TUNEL and immunofluorescence co-localization assay, we observed Rb1 also inhibited endogenous reactive oxygen species (ROS), tightly associated with increased superoxide dismutase (SOD), catalase (CAT) levels, and decreased malondialdehyde (MDA). Subsequently, the silent information regulator Sirtuni1(SIRT1)/peroxisome proliferator-activated receptor-γ coactivator α (PGC-1α) pathway were activated, whereas, phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were alleviated. All of which led to mitochondria dysfunction via promoting mitochondrial fusion protein Mitofusion 1 (MFN1) and inhibiting dynamin-related protein 1 (DRP1) expression and apoptosis in lungs. In BEAS-2B cells, Rb1 played a similar role as SIRT1 agonist (SRT1720), including mitochondrial membrane potential enhancement, mitochondrial ROS and DRP1 translocation to mitochondria decrease. Our findings suggest that Rb1 maintains mitochondria integrity by activating SIRT1/PGC-1α, inhibiting PI3K/AKT, thereby ameliorates asthmatic airway inflammation.\u003c/p\u003e","manuscriptTitle":"Ginsenoside Rb1 alleviates airway inflammation in asthma by regulating mitochondrial dysfunction through SIRT1/PGC-1α and PI3K/AKT signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-19 15:25:12","doi":"10.21203/rs.3.rs-3957667/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":"9b4b3c21-5d77-4f83-8922-8c0c5e9fd103","owner":[],"postedDate":"March 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-29T03:18:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-19 15:25:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3957667","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3957667","identity":"rs-3957667","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}