Atractylenolide II combined with Interferon-γ synergistically ameliorates colorectal cancer progression in vivo and in vitro by blocking the NF-kB p65/PD-L1 pathway.

Purpose: Atractylodes macrocephala Koidz is a widely used classical traditional Chinese herbal medicine, that has shown remarkable efficacy in cancers. Colorectal cancer (CRC) is the most common malignant tumor globally. Interferon (IFN)-γ, a prominent cytokine involved in anti-tumor immunity that has cytostatic, pro-apoptotic, and immune-stimulatory properties for the detection and removal of transformed cells. Atractylenolides-II (AT-II) belongs to the lactone compound that is derived from Atractylodes macrocephala Koidz with anti-cancer activity. However, whether AT-II combined with IFN-γ modulates CRC progression and the underlying mechanisms remain unclear. The present study aimed to elucidate the efficacy and pharmaceutical mechanism of action of AT-II combined with IFN-γ synergistically against CRC by regulating the NF-kB p65/PD-L1 signaling pathway. Methods: HT29 and HCT15 cells were treated with AT-II and IFN-γ alone or in combination and cell viability, migration, and invasion were then analyzed using Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. Furthermore, the underlying mechanism was investigated through western blot assay. The role of AT-II combined with IFN-γ on tumor growth and lung metastases was estimated in vivo. Finally, the population of lymphocytes in tumor tissues of lung metastatic C57BL/6 mice and the plasma cytokine levels were confirmed by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Results: AT-II or the combination IFN-γ significantly inhibited the growth and migration abilities of CRC cells in vitro and in vivo. The biological mechanisms behind the beneficial effects of AT-II combined with IFN-γ were also measured and inhibition of p38 MAPK, FAK, Wnt/ß-catenin, Smad, and NF-kB p65/PD-L1 pathways was observed. Moreover, AT-II combined with IFN-γ significantly inhibited HCT15 xenograft tumor growth and lung metastases in C57BL/6 mice, which was accompanied by lymphocyte infiltration into the tumor tissues and inflammatory response inactivation. Conclusions: The results showed that the AT-II in combination with IFN-γ could be used as a potential strategy for tumor immunotherapy in CRC. More importantly, the mechanism by which AT-II suppressed CRC progressions was by inhibiting the NF-kB p65/PD-L1 signal pathway.

Pharmacokinetic study and neuropharmacological effects of atractylenolide â ¢ to improve cognitive impairment via PI3K/AKT/GSK3ß pathway in intracerebroventricular-streptozotocin rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese herbal remedy Atractylodes macrocephala Koidz is renowned for its purported gastrointestinal regulatory properties and immune-enhancing capabilities. Atractylenolide III (ATL III), a prominent bioactive compound in Atractylodes macrocephala Koidz, has demonstrated significant pharmacological activities. However, its impact on neuroinflammation, oxidative stress, and therapeutic potential concerning Alzheimer's disease (AD) remain inadequately investigated. AIM OF THE STUDY: This study aims to assess the plasma pharmacokinetics of ATL III in Sprague-Dawley (SD) rats and elucidate its neuropharmacological effects on AD via the PI3K/AKT/GSK3ß pathway. Through this research, we endeavor to furnish experimental substantiation for the advancement of novel therapeutics centered on ATL III. MATERIALS AND METHODS: The pharmacokinetic profile of ATL III in SD rat plasma was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). AD models were induced in SD rats through bilateral intracerebroventricular (ICV) administration of streptozotocin (STZ). ATL III was administered at doses of 0.6 mg/kg, 1.2 mg/kg, and 2.4 mg/kg, while donepezil (1 mg/kg) served as control. Cognitive function assessments were conducted employing behavioral tests including the Morris Water Maze and Novel Object Recognition. Neuronal pathology and histological changes were evaluated through Nissl staining and Hematoxylin-Eosin (HE) staining, respectively. Oxidative stress levels were determined by quantifying malondialdehyde (MDA) content and total superoxide dismutase (T-SOD) activity. Molecular docking analysis was employed to explore the direct binding between ATL III and its relevant targets, followed by validation using Western blot (WB) experiments to assess the expression of p-Tau, PI3K, AKT, GSK3ß, and their phosphorylated forms. RESULTS: Within the concentration range of 5-500 ng/mL, ATL III demonstrated exceptional linearity (R2 = 0.9991), with a quantification limit of 5 ng/mL. In male SD rats, ATL III exhibited a Tmax of 45 min, a t1/2 of 172.1 min, a Cmax of 1211 ng/L, and an AUC(0-t) of 156031 ng/L*min. Treatment with ATL III significantly attenuated Tau hyperphosphorylation in intracerebroventricular-streptozotocin (ICV-STZ) rats. Furthermore, ATL III administration mitigated neuroinflammation and oxidative stress, as evidenced by reduced Nissl body loss, alleviated histological alterations, decreased MDA content, and enhanced T-SOD activity. Molecular docking analyses revealed strong binding affinity between ATL III and the target genes PI3K, AKT, and GSK3ß. Experimental validation corroborated that ATL III stimulated the phosphorylation of PI3K and AKT while reducing the phosphorylation of GSK3ß. CONCLUSIONS: Our results indicate that ATL III can mitigate Tau protein phosphorylation through modulation of the PI3K/AKT/GSK3ß pathway. This attenuation consequently ameliorates neuroinflammation and oxidative stress, leading to enhanced learning and memory abilities in ICV-STZ rats.

Atractylenolide I ameliorates sepsis-induced cardiomyocyte injury by inhibiting macrophage polarization through the modulation of the PARP1/NLRP3 signaling pathway.

Sepsis-induced cardiomyopathy (SIC) leads to high mortality and has no effective treatment strategy. Atractylenolide Ⅰ (AT-I) is a sesquiterpene lactone compound and possesses various biological activities such as anti-inflammatory and organ protection. This study was designed to explore the role and the mechanism of AT-I in SIC. CCK-8 assay was used to assess the viability of AT-I-treated RAW 264.7 cells and immunofluorescence assay was used to detect M1 marker CD86. The expressions of M1 markers Cox2, iNOS and CD11b and PARP1/NLRP3 signaling pathway-related proteins were detected using western blot. The transfection efficiency of oe-PARP1 was examined with RT-qPCR and western blot. The ROS activity in H9c2 cells was detected using DCFH-DA assay and western blot was used to detect the expressions of inflammation- and oxidative stress-related proteins. The apoptosis of H9c2 cells was detected using flow cytometry and western blot. The present study found that AT-I inhibited LPS-induced M1 polarization in RAW 264.7 cells through the downregulation of PARP1/NLRP3 signaling pathway, thereby inhibiting the oxidative stress and apoptosis of H9c2 cells. In conclusion, AT-I might be a promising therapeutic agent for SIC by suppressing macrophage polarization through the modulation of PARP1/NLRP3 signaling pathway.

Atractylenolide I Alleviates Indomethacin-Induced Gastric Ulcers in Rats by Inhibiting NLRP3 Inflammasome Activation.

Atractylodes macrocephala Koidz, a traditional Chinese medicine, contains atractylenolide I (ATR-I), which has potential anticancer, anti-inflammatory, and immune-modulating properties. This study evaluated the therapeutic potential of ATR-I for indomethacin (IND)-induced gastric mucosal lesions and its underlying mechanisms. Noticeable improvements were observed in the histological morphology and ultrastructures of the rat gastric mucosa after ATR-I treatment. There was improved blood flow, a significant decrease in the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1ß, and IL-18, and a marked increase in prostaglandin E2 (PGE2) expression in ATR-I-treated rats. Furthermore, there was a significant decrease in the mRNA and protein expression levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cysteinyl aspartate specific proteinase-1 (caspase-1), and nuclear factor-κB (NF-κB) in rats treated with ATR-I. The results show that ATR-I inhibits the NLRP3 inflammasome signaling pathway and effectively alleviates local inflammation, thereby improving the therapeutic outcomes against IND-induced gastric ulcers in rats.

Chemopreventive effects of atractylenolide-III on mammary tumorigenesis via activation of the Nrf2/ARE pathway through autophagic degradation of Keap1.

The incidence of breast cancer is increasing annually, making it a major health threat for women. Chemoprevention using natural, dietary, or synthetic products has emerged as a promising approach to address this growing burden. Atractylenolide-III (AT-III), a sesquiterpenoid present in various medicinal herbs, has demonstrated potential therapeutic effects against several diseases, including tumors, nonalcoholic fatty liver disease, and cerebral ischemic injury. However, its impact on breast cancer chemoprevention remains unexplored. In this study, we used an N-methyl-N-nitrosourea (NMU)-induced rat breast cancer model and 17ß-estradiol (E2)-treated MCF-10A cells to evaluate the chemopreventive potential of AT-III on mammary tumorigenesis. AT-III inhibited mammary tumor progression, evidenced by reduced tumor volume and multiplicity, prolonged tumor latency, and the reversal of NMU-induced weight loss. Furthermore, AT-III suppressed NMU-induced inflammation and oxidative stress through the Nrf2/ARE pathway in breast cancer tissues. In vitro, AT-III effectively suppressed E2-induced anchorage-independent growth and cell migration in MCF-10A cells. Nrf2 knockdown attenuated the protective effects of AT-III, highlighting the pivotal role of Nrf2 in AT-III-mediated suppression of tumorigenesis. The mechanism involves the induction of Nrf2 expression by AT-III through the autophagic degradation of Kelch-like ECH-associated protein 1 (Keap1). Overall, the results of this study indicate that AT-III is a promising candidate for breast cancer chemoprevention and provide valuable insights into its molecular interactions and signaling pathways.

Effect of atractylenolide III on zearalenone-induced Snail1-mediated epithelial-mesenchymal transition in porcine intestinal epithelium.

BACKGROUND: The intestinal epithelium performs essential physiological functions, such as nutrient absorption, and acts as a barrier to prevent the entry of harmful substances. Mycotoxins are prevalent contaminants found in animal feed that exert harmful effects on the health of livestock. Zearalenone (ZEA) is produced by the Fusarium genus and induces gastrointestinal dysfunction and disrupts the health and immune system of animals. Here, we evaluated the molecular mechanisms that regulate the effects of ZEA on the porcine intestinal epithelium. RESULTS: Treatment of IPEC-J2 cells with ZEA decreased the expression of E-cadherin and increased the expression of Snai1 and Vimentin, which induced Snail1-mediated epithelial-to-mesenchymal transition (EMT). In addition, ZEA induces Snail-mediated EMT through the activation of TGF-ß signaling. The treatment of IPEC-J2 cells with atractylenolide III, which were exposed to ZEA, alleviated EMT. CONCLUSIONS: Our findings provide insights into the molecular mechanisms of ZEA toxicity in porcine intestinal epithelial cells and ways to mitigate it.

Atractylenolide-I Alleviates Hyperglycemia-Induced Heart Developmental Malformations through Direct and Indirect Modulation of the STAT3 Pathway.

BACKGROUND: Gestational diabetes could elevate the risk of congenital heart defects (CHD) in infants, and effective preventive and therapeutic medications are currently lacking. Atractylenolide-I (AT-I) is the active ingredient of Atractylodes Macrocephala Koidz (known as Baizhu in China), which is a traditional pregnancy-supporting Chinese herb. PURPOSE: In this study, we investigated the protective effect of AT-I on the development of CHD in embryos exposed to high glucose (HG). STUDY DESIGN AND METHODS: First, systematic review search results revealed associations between gestational diabetes mellitus (GDM) and cardiovascular malformations. Subsequently, a second systematic review indicated that heart malformations were consistently associated with oxidative stress and cell apoptosis. We assessed the cytotoxic impacts of Atractylenolide compounds (AT-I, AT-II, and AT-III) on H9c2 cells and chick embryos, determining an optimal concentration of AT-I for further investigation. Second, immunofluorescence, western blot, Polymerase Chain Reaction (PCR), and flow cytometry were utilized to delve into the mechanisms through which AT-I mitigates oxidative stress and apoptosis in cardiac cells. Molecular docking was employed to investigate whether AT-I exerts cardioprotective effects via the STAT3 pathway. Then, we developed a streptozotocin-induced diabetes mellitus (PGDM) mouse model to evaluate AT-I's protective efficacy in mammals. Finally, we explored how AT-I protects hyperglycemia-induced abnormal fetal heart development through microbiota analysis and untargeted metabolomics analysis. RESULTS: The study showed the protective effect of AT-I on embryonic development using a chick embryo model which rescued the increase in the reactive oxygen species (ROS) and decrease in cell survival induced by HG. We also provided evidence suggesting that AT-I might directly interact with STAT3, inhibiting its phosphorylation. Further, in the PGDM mouse model, we observed that AT-I not only partially alleviated PGDM-related blood glucose issues and complications but also mitigated hyperglycemia-induced abnormal fetal heart development in pregnant mice. This effect is hypothesized to be mediated through alterations in gut microbiota composition. We proposed that dysregulation in microbiota metabolism could influence the downstream STAT3 signaling pathway via EGFR, consequently impacting cardiac development and formation. CONCLUSIONS: This study marks the first documented instance of AT-I's effectiveness in reducing the risk of early cardiac developmental anomalies in fetuses affected by gestational diabetes. AT-I achieves this by inhibiting the STAT3 pathway activated by ROS during gestational diabetes, significantly reducing the risk of fetal cardiac abnormalities. Notably, AT-I also indirectly safeguards normal fetal cardiac development by influencing the maternal gut microbiota and suppressing the EGFR/STAT3 pathway.

Atractylenolide II regulates the proliferation, ferroptosis, and immune escape of hepatocellular carcinoma cells by inactivating the TRAF6/NF-κB pathway.

Hepatocellular carcinoma (HCC) is a common and lethal tumor worldwide. Atractylenolide II (AT-II) is a natural sesquiterpenoid monomer, with anti-tumor effect. To address the effect and mechanisms of AT-II on HCC. The role and mechanisms of AT-II were assessed through cell counting kit-8, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescence, and western blot experiments in Hep3B and Huh7 cells. In vivo experiments were conducted in BALB/c nude mice using immunohistochemistry and western blot assays. AT-II decreased the cell viability of Hep3B and Huh7 cells with a IC50 of 96.43 µM and 118.38 µM, respectively. AT-II increased relative Fe2+ level, which was further promoted with the incubation of erastin and declined with the ferrostatin-1 in Hep3B and Huh7 cells. AT-II enhanced the level of ROS and MDA, but reduced the GSH level, and the expression of xCT and GPX4. AT-II elevated the percent of CD8+ T cells and the IFN-γ contents, and declined the IL-10 concentrations and the expression of PD-L1 in Hep3B and Huh7 cells. AT-II downregulated the relative protein level of TRAF6, p-p65/p-65, and p-IkBα/IkBα, which was rescued with overexpression of TRAF6. Upregulation of TRAF6 also reversed the effect of AT-II on proliferation, ferroptosis, and immune escape in Hep3B cells. In vivo, AT-II reduced tumor volume and weight, the level of GPX4, xCT, and PD-L1, and the expression of TRAF6, p-p65/p-65, and p-IkBα/IkBα, with the increased expression of CD8. AT-II modulated the proliferation, ferroptosis, and immune escape of HCC cells by downregulating the TRAF6/NF-κB pathway.

Treating cancer through modulating exosomal protein loading and function: The prospects of natural products and traditional Chinese medicine.

Exosomes, small yet vital extracellular vesicles, play an integral role in intercellular communication. They transport critical components, such as proteins, lipid bilayers, DNA, RNA, and glycans, to target cells. These vesicles are crucial in modulating the extracellular matrix and orchestrating signal transduction processes. In oncology, exosomes are pivotal in tumor growth, metastasis, drug resistance, and immune modulation within the tumor microenvironment. Exosomal proteins, noted for their stability and specificity, have garnered widespread attention. This review delves into the mechanisms of exosomal protein loading and their impact on tumor development, with a focus on the regulatory effects of natural products and traditional Chinese medicine on exosomal protein loading and function. These insights not only offer new strategies and methodologies for cancer treatment but also provide scientific bases and directions for future clinical applications.

Atractylenolide II Suppresses Glycolysis and Induces Apoptosis by Blocking the PADI3-ERK Signaling Pathway in Endometrial Cancer Cells.

Atractylenolide II (AT-II), the major bioactive compound of Atractylodes macrocephala, exhibits anti-cancer activity against many types of tumors, but the roles and the potential mechanisms in endometrial cancer remain unclear. In the present study, AT-II treatment was found to significantly suppress RL95-2 and AN3CA cell proliferation and glycolysis, and induced their apoptosis by inactivating the ERK signaling pathway, accompanied by the changing expression of the glycolytic key enzymes and apoptotic-related proteins. Peptidyl arginine deiminase 3 (PADI3), as the candidate target gene of AT-II, was highly expressed in the endometrial cancer tissues and associated with a poor prognosis according to bioinformatics analysis. PADI3 knockdown inhibited proliferation and glycolysis in endometrial cancer cells and induced cell apoptosis. Furthermore, AT-II negatively regulated the expression of PADI3, and PADI3 overexpression reversed the effects of AT-II on endometrial cancer cells. Our findings suggested that the anti-cancer function of AT-II is associated with the suppression of glycolysis and induction of apoptosis by blocking the PADI3-ERK signaling pathway. Thus, AT-II represents a novel therapeutic target for endometrial cancer and targeting AT-II may serve as a potential strategy for the clinical therapy of endometrial cancer.

Atractylenolide I Suppresses A1 Astrocyte Activation to Improve Depression in Mice.

This work aimed to investigate the role of atractylenolide I (ATR) in resisting depression and its mechanism of action. The mouse model of depression was constructed through chronic unpredictable mild stress (CUMS) method. After ATR intervention, changes in the depression-related behaviors of mice were detected through open field test and elevated plus maze. In addition, enzyme-linked immunosorbent assay (ELISA) was conducted to detect inflammatory factor levels. Real-time fluorescence quantitative PCR (RT-qPCR) was performed to measure the mRNA levels of A1/A2 astrocyte markers. Furthermore, primary astrocytes were induced in vitro, and the A1 differentiation level was detected by ELISA and RT-qPCR assays. ATR improved the behaviors of CUMS mice and alleviated the depression symptoms. Moreover, it reduced tissue inflammation, inhibited the A1 differentiation of astrocytes, and decreased the mRNA levels of A1 markers. After NLRP3 knockout, the effects of ATR were suppressed. Similarly, in vitro experimental results also revealed that ATR suppressed the A1 differentiation of astrocytes. Based on molecular dynamics and small molecule-protein docking results, ATR mainly targeted NLRP3 and suppressed the NLRP3-mediated A1 differentiation. We discover that ATR can target NLRP3 to suppress A1 differentiation of astrocytes, restrain tissue inflammation, and improve the depression symptoms in mice.

Atractylenolide-III attenuates osteoarthritis by repolarizing macrophages through inactivating TLR4/NF-κB signaling.

BACKGROUND: As a common chronic musculoskeletal condition, osteoarthritis (OA) presently lacks particular treatment strategies. The aim of this study was to examine how AT-III therapies affected macrophage repolarity in order to slow down the advancement of OA. METHODS: RAW264.7 macrophages were polarized to M1 subtypes then administered with different concentrations of AT-III. Immunofluorescence, qRT-PCR and flow cytometry were used to assess the polarization of the macrophages. The mechanism of AT-III repolarize macrophages was evaluated by western blot. Furthermore, the effects of macrophage conditioned media (CM) on the migration, proliferation, and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were investigated using CCK-8 assays, the scratch test, and alcian blue staining. The effects of macrophage CM on chondrocyte proliferation and degeneration were investigated using CCK-8 and qRT-PCR. In vivo micro-CT and histological observations were performed on rats with anterior cruciate ligament transection and partial medial meniscectomy, either with or without AT-III treatment. RESULTS: AT-III repolarized M1 macrophages to M2 phenotype. Mechanistically, AT-III reduced the expression of Toll-like receptor(TLR) 4 induced by lipopolysaccharide in RAW264.7 and lowered nuclear factor-κB (NF-κB) signaling molecules p-p65 and p-IκBα. The TLR4 agonist RS09 reversed the effects of AT-III on macrophage repolarization. AT-III-induced macrophages CM stimulated BMSCs migration, proliferation and chondrogenic differentiation. AT-III-treated macrophage CM promoted chondrocyte proliferation while inhibiting chondrocyte degeneration. In vivo, AT-III treatment alleviated the degree of synovitis, inhibited subchondral bone remodeling and reduced cartilage destruction in the rat OA model. CONCLUSIONS: AT-III attenuates OA by repolarizing macrophages through inactivating TLR4/NF-κB signaling. These data suggest that AT-III may be an effective therapeutic candidate for OA treatment.

Atractylenolide I improves behaviors in mice with depression-like phenotype by modulating neurotransmitter balance via 5-HT2A.

To explore the antidepressant effects and targets of atractylenolide I (ATR) through a network pharmacological approach. Relevant targets of ATR and depression analyzed by network pharmacology were scored (identifying 5-HT2A targets). Through elevated plus maze, open field, tail suspension, and forced swimming tests, the behavioral changes of mice with depression (chronic unpredictable mild stress [CUMS]) were examined, and the levels of neurotransmitters including serotonin, dopamine, and norepinephrine (5-HT, DA, and NE) were determined. The binding of ATR to 5-HT2A was verified by small molecular-protein docking. ATR improved the behaviors of CUMS mice, elevated their levels of neurotransmitters 5-HT, DA, and NE, and exerted a protective effect on their nerve cell injury. After 5-HT2A knockout, ATR failed to further improve the CUMS behaviors. According to the results of small molecular-protein docking and network pharmacological analysis, ATR acted as an inhibitor by binding to 5-HT2A. ATR can improve the behaviors and modulate the neurotransmitters of CUMS mice by targeting 5-HT2A.

Atractylenolide II ameliorates myocardial fibrosis and oxidative stress in spontaneous hypertension rats.

BACKGROUND: Hypertension is a well-recognized risk factor for cardiovascular, which is also a critical factor in causing myocardial fibrosis (MF). OBJECTIVE: The study aimed to explore the effect of Atractylenolide II (ATL-II) on MF and oxidative stress in spontaneous hypertension rats (SHR). METHODS: The body weight of rats after injection of ATL-II was quantitatively analyzed. The left ventricular function of SHR was evaluated by Echocardiographic. HE staining, Masson trichrome staining, left ventricular mass index (LVMI) and immunofluorescence was applied to investigate the effects of ATL-II on MF. RT qPCR was used to detect the Collagen I, α-SMA, Fibronectin, and Vimentin mRNA expression levels in myocardial slices. The effect ATL-II on cardiomyocyte apoptosis was detected by TUNEL staining and western blot. An immunohistochemistry assay was conducted to detect α-SMA protein and TGF-ß1 protein. The contents of H2O2, GSH-PX, SOD, and MDA were measured by colorimetry. RESULTS: ATL-II could dose-dependently improve the BW of SHRs (P< 0.05) and enhance myocardial function. Moreover, ATL-II effectively reduced cardiomyocyte apoptosis in SHRs. Alternatively, ATL-II could inhibit the Collagen I, α-SMA, Fibronectin, and Vimentin mRNA and protein expression levels in SHRs. ATL-II could ameliorate oxidative stress by improving the activities of SOD and GSH-PX and lowering the contents of H2O2 and MDA in ATL-II-treated SHRs, which reach about 80%. CONCLUSION: ATL-II could exert an inhibiting effect on MF and oxidative stress in SHRs. Hence, ATL-II may hold promise for the treatment of MF and oxidative stress in Spontaneous Hypertension.

Effect of Atractylenolide Ⅰ on Lung Injury in Rats with Recurrent Respiratory Tract Infection of Lung and Spleen Qi Deficiency by Regulating the PI3K/Akt/mTOR Signaling Pathway / 中药新药与临床药理

Objective To investigate the effect of atractylenolideⅠon lung injury in rats with recurrent respiratory tract infection(RRTI)of lung and spleen qi deficiency by regulating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway.Methods Eighty-four rats were randomly separated into a control group,a model group,a low-dose atractylenolideⅠgroup,a high-dose atractylenolideⅠgroup,a positive drug group,an insulin-like growth factor-1(IGF-1)group,and a high-dose atractylenolide Ⅰ+IGF-1 group,with 12 rats in each group.Except for the control group,the RRTI rat model of lung and spleen qi deficiency was constructed using a combination of fatigue,dietary disorders,and fumigation method with shavings and tobacco among rats in other groups.After the model is successfully copied,the model was administered once a day for 6 weeks.Animal lung function instrument was applied to detect the changes of peak expiratory flow(PEF),forced expiratory volume in first second(FEV1),forced vital capacity(FVC)in rats.The changes of wet/dry mass ratio of lungs in rats were detected.HE staining was applied to detect pathological changes of lung tissue in rats of each group.ELISA was applied to detect the levels of interleukin(IL)-6,tumor necrosis factor-α(TNF-α),malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)in rat lung tissue.Western Blot was applied to determine the protein expressions of p-PI3K,p-Akt,and p-mTOR in rat lung tissue.Results Compared with the control group,rats in the model group showed a decrease in PEF,FEV1 and FVC(P＜0.01)and an increase in the wet/dry mass ratio of lungs(P＜0.01).The alveolar septa in lung tissues had become larger.Pulmonary interstitial edema and a large amount of inflammatory cell infiltration were found.The levels of IL-6,TNF-α and MDA in lung tissue increased(P＜0.01),and the SOD activity decreased(P＜0.01).The protein expressions of p-PI3K,p-Akt,and p-mTOR in lung tissue increased(P＜0.01).Compared with the model group,rats in the low-,high-dose atractylenolideⅠgroups,and positive drug group showed an increase in PEF,FEV1,and FVC,and a decrease in the wet/dry mass ratio of lungs(P＜0.01).Pathologic damage in lung tissue was alleviated.The levels of IL-6,TNF-α,MDA decreased and SOD activity in lung tissue increased(P＜0.01).The protein expressions of p-PI3K,p-Akt,and p-mTOR in lung tissue decreased(P＜0.01),while the corresponding indicators in the IGF-1 group showed opposite trends(P＜0.01).Compared with the high-dose group of atractylenolide I,rats in the high-dose atractylenolide I+IGF-1 group showed a decrease in PEF,FEV1 and FVC,and an increase in the wet/dry mass ratio of lungs(P＜0.01).Pathologic damage in lung tissue was increased.The levels of IL-6,TNF-α,MDA increased and the SOD activity in lung tissue decreased(P＜0.01).The protein expressions of p-PI3K,p-Akt,and p-mTOR in lung tissue increased(P＜0.05,P＜0.01).Conclusion AtractylenolideⅠmay improve lung injury in RRTI rats of lung and spleen qi deficiency by inhibiting the PI3K/Akt/mTOR pathway.

Atractylenolide III ameliorated reflux esophagitis via PI3K/AKT/NF-κB/iNOS pathway in rats.

Reflux esophagitis (RE), an esophageal inflammation caused by reflux of gastric contents, often damages the lower esophagus, seriously affecting the quality of life of patients. This study aims to investigate the therapeutic effects and underlying molecular mechanisms of atractylenolide III (ATL III) on RE model rats. In this research, the RE rat model is established sequentially following hemipyloric ligation, cardia transection, and hydrochloric acid perfusion. Further, the RE-induced rats are intragastrically administrated with ATL III (0.6, 1.2, and 2.4 mg/kg/D) for 28 days to evaluate ATL III therapeutic effects. To study the molecular mechanism, RE rats are treated with a phosphoinositide-3 kinase (PI3K) agonist (740 Y-P) combined with ATL III. The histopathological changes in the esophagus are eventually observed by hematoxylin & eosin (H&E) staining. In addition to changes in gastric pH and levels of reactive oxygen species (ROS), enzyme-linked immunosorbent assay (ELISA) and Western blot analyses are used to detect the expression levels of tumor necrosis factor-α (TNF-α, mmol/L), interleukin (IL)-8, IL-6, IL-1ß in the esophageal tissues. As a result, the lesions in the esophageal tissues of RE rats are alleviated, decreasing the macroscopic observation scores of the esophageal mucosa after ATL III treatment,. The experimental results indicated significantly increased pH value of the gastric contents and reduced ROS, thiobarbituric acid reactants (TBARS), TNF-α, IL-8, IL-6, and IL-1ß levels, as well as expression levels of p-PI3K, p-AKT, iNOS, and nuclear NF-κB proteins in esophageal tissues. In conclusion, the study indicated that ATL III could efficiently treat RE in rats by inhibiting oxidative stress and inflammatory damage through the PI3K/AKT/NF-κB/iNOS pathway.

Atractylenolide III Ameliorates Bile Duct Ligation-Induced Liver Fibrosis by Inhibiting the PI3K/AKT Pathway and Regulating Glutamine Metabolism.

Liver fibrosis is one of the leading causes of hepatic sclerosis and hepatocellular carcinoma worldwide. However, the complex pathophysiological mechanisms of liver fibrosis are unknown, and no specific drugs are available to treat liver fibrosis. Atractylenolide III (ATL III) is a natural compound isolated from the plant Atractylodes lancea (Thunb.) DC. that possesses antioxidant properties and the ability to inhibit inflammatory responses. In this study, cholestatic hepatic fibrosis was induced in mice using a bile duct ligation (BDL) model and treated with 10 mg/kg and 50 mg/kg of ATL III via gavage for 14 days. ATL III significantly reduced the liver index, lowered serum ALT and AST levels, and reduced liver injury in bile-duct-ligated mice. In addition, ATL III significantly attenuated histopathological changes and reduced collagen deposition. ATL III reduced the expression of fibrosis-related genes α-smooth muscle actin (α-SMA), Collagen I (col1a1), Collagen IV (col4a2), and fibrosis-related proteins α-SMA and col1a1 in liver tissue. Using RNA sequencing (RNA-seq) to screen molecular targets and pathways, ATL III was found to affect the PI3K/AKT singling pathway by inhibiting the phosphorylation of PI3K and AKT, thereby ameliorating BDL-induced liver fibrosis. Gas chromatography-mass spectrometry (GC-MS) was used to evaluate the effect of ATL III on liver metabolites in BDL mice. ATL III further affected glutamine metabolism by down-regulating the activity of glutamine (GLS1) and glutamine metabolism. ATL III further affected glutamine metabolism by down-regulating the activity of glutaminase (GLS1), as well as glutamine metabolism. Therefore, we conclude that ATL III attenuates liver fibrosis by inhibiting the PI3K/AKT pathway and glutamine metabolism, suggesting that ATL III is a potential drug candidate for treating liver fibrosis.

Atractylenolide-III alleviates osteoarthritis and chondrocyte senescence by targeting NF-κB signaling.

Atractylenolide-III (AT-III) is well known as its role in antioxidant and anti-inflammatory. Present study was aimed to figure out its effects on osteoarthritis and potential mechanisms. Rat model, human osteoarthritis cartilage explants as well as rat/human chondrocyte cultures were prepared to test AT-III's effects on osteoarthritis progression and chondrocyte senescence. Potential targeted molecules of AT-III were predicted using network pharmacology and molecular docking, assessed by Western blotting and then verified with rescue experiments. AT-III treatment alleviated osteoarthritis severity (shown by OARSI grading score and micro-CT) and chondrocyte senescence (indexed by levels of SA-ß-gal, P16, P53, MMP13, ROS and ratio of healthy/collapsed mitochondrial membrane potentials). Network pharmacology and molecular docking suggested that AT-III might play role through NF-κB pathway. Further experiments revealed that AT-III reduced phosphorylation of IKKα/ß, IκBα and P65 in NF-κB pathway. As well as nuclear translocation of p65. Both in vivo and in vitro experiments indicated that AT-III's effects on osteoarthritis and anti-senescence were reversed by an NF-κB agonist. AT-III could alleviate osteoarthritis by inhibiting chondrocyte senescence through NF-κB pathway, which indicated that AT-III is a prospective drug for osteoarthritis treatment.

Paeoniflorin, ferulic acid, and atractylenolide III improved LPS-induced neuroinflammation of BV2 microglia cells by enhancing autophagy.

Microglia hyperactivation is an important cause of neuroinflammation in Alzheimer's disease (AD). Paeoniflorin (PF), ferulic acid (FA), and atractylenolide III (ATL) are potent in anti-inflammation and neuroprotection. Multiple components can act on different targets simultaneously to exert synergistic therapeutic effects and exploring the synergistic potential between compounds is an important area of research. We investigated the effects of PF, FA, and ATL, alone or in combination, on LPS-induced neuroinflammation and autophagy in BV2 microglia cells. We found that PF, FA, and ATL, alone or in combination, significantly reduced the production of inflammatory factors such as IL-6, IL-1ß, and TNF-α, especially in the PF + FA + ATL group, which performed the best. In addition, the combination of PF, FA, and ATL significantly increased the expression of autophagy-related proteins p-AMPK, p-ULK1, Beclin1, LC3, and TFEB and decreased the expression of p62. Moreover, the restoration of autophagic flux by the combination of PF, FA, and ATL was abrogated by the addition of the autophagy inhibitor Wortmannin. In conclusion, PF, FA, and ATL have a synergistic effect in reducing LPS-induced inflammatory factor release from BV2 microglia cells, and its protective effect may be through activation of the AMPK/ULK1/TFEB autophagic signaling pathway.

Atractylenolide III Ameliorated Autophagy Dysfunction via Epidermal Growth Factor Receptor-Mammalian Target of Rapamycin Signals and Alleviated Silicosis Fibrosis in Mice.

Atractylenolide III (ATL-III) is a major active constituent of the natural plant Atractylodes rhizome. Our previous study has shown that ATL-III may alleviate alveolar macrophage apoptosis via the inhibition of the mammalian target of rapamycin (mTOR)-mediated autophagy of human silicosis. Therefore, we aimed to further explore the function of ATL-III in autophagy, apoptosis, and pulmonary fibrosis by establishing the ATL-III-intervened silicosis mouse model in this study. Meanwhile, we sought and then verified potential autophagy-related signaling pathways by matching differentially expressed genes (attained by RNA sequencing) and the autophagy database. In this study, RNA-sequencing results implied that the epidermal growth factor receptor, the crucial upstream activator of mTOR, was seen as a potential autophagy-regulatory molecule in the ATL-III-intervened silicosis mouse model. The finding of this study was that ATL-III might improve the disorder of autophagic degradation via the activation of epidermal growth factor receptor-mTOR signals in the pulmonary tissue of the silicosis mouse model. ATL-III also alleviated cell apoptosis and silicotic fibrosis. Overall, we supposed that ATL-III might be a potential protective medicine, which had a regulatory effect on autophagy, for the intervention of silicotic fibrosis. In the future, the therapeutic drugs for silicosis should be further focused on the development and application of such natural autophagy agents.