Costunolide Inhibits Chronic Kidney Disease Development by Attenuating IKKß/NF-κB Pathway.

Background: Chronic kidney disease (CKD) is a significant worldwide health concern that leads to high mortality rates. The bioactive substance costunolide (CTD) has demonstrated several pharmacological effects and holds promise as a CKD treatment. This study aims to investigate the impact of CTD on CKD and delve into its mechanisms of action. Methods: Unilateral ureteral obstruction (UUO) methods and renal fibrosis mice models were created. Various concentrations of CTD were injected into UUO mice models to investigate the therapeutic effects of CTD on renal fibrosis of mice. Then, renal morphology, pathological changes, and the expression of genes related to fibrosis, inflammation and ferroptosis were analysed. RNA sequencing was utilized to identify the main biological processes and pathways involved in renal injury. Finally, both overexpression and inhibition of IKKß were studied to examine their respective effects on fibrosis and inflammation in both in vitro and in vivo models. Results: CTD treatment was found to significantly alleviate fibrosis, inflammation and ferroptosis in UUO-induced renal fibrosis mice models. The results of RNA sequencing suggested that the IKKß acted as key regulatory factor in renal injury and the expression of IKKß was increased in vitro and in vivo renal fibrosis model. Functionally, down-regulated IKKß expression inhibits ferroptosis, inflammatory cytokine production and collagen deposition. Conversely, IKKß overexpression exacerbates progressive renal fibrosis. Mechanistically, CTD alleviated renal fibrosis and inflammation by inhibiting the expression of IKKß and attenuating IKKß/NF-κB pathway. Conclusion: This study demonstrates that CTD could mitigate renal fibrosis, ferroptosis and inflammation in CKD by modulating the IKKß/NF-κB pathway, which indicates targeting IKKß has an enormous potential for treating CKD.

Costunolide ameliorates MNNG-induced chronic atrophic gastritis through inhibiting oxidative stress and DNA damage via activation of Nrf2.

BACKGROUND: Chronic atrophic gastritis (CAG) is a chronic digestive disease. Modern research has revealed substantial evidence indicating that the progression of CAG is closely linked to the occurrence of oxidative stress-induced DNA damage and apoptosis in the gastric mucosa. Additionally, research has indicated that Costunolide (COS), the primary active compound found in Aucklandiae Radix, a traditional herb, exhibits antioxidant properties. Nevertheless, the therapeutic potential of COS in treating CAG and its molecular targets have not yet been determined. PURPOSE: The objective of this research was to explore the potential gastric mucosal protective effects and mechanisms of COS against N-Methyl-N´-nitro-N-nitrosoguanidine (MNNG)-induced CAG. METHODS: Firstly, the MNNG-induced rat CAG model was established in vivo. Occurrence of CAG was detected through macroscopic examination of the stomachs and H&E staining. Additionally, we assessed oxidative stress, DNA damage, and apoptosis using biochemical detection, Western blot, immunohistochemistry and immunofluorescence. Then, an in vitro model was developed to induce MNNG-induced damage in GES-1 cells, and the occurrence of cell damage was determined by Hoechst 33,342 staining and flow cytometry. Finally, the key targets of COS for the treatment of CAG were identified through molecular docking, cellular thermal shift assay (CETSA), and inhibitor ML385. RESULTS: In vivo studies demonstrated that COS promotes the expression of Nrf2 in gastric tissues. This led to an increased expression of SOD, GSH, HO-1, while reducing the production of MDA. Furthermore, COS inhibited DNA damage and apoptosis by suppressing the expression of γH2AX and PARP1 in gastric tissues. In vitro studies showed that COS effectively reversed apoptosis induced by MNNG in GES-1 cells. Additionally, COS interacted with Nrf2 to promote its expression. Furthermore, the expression levels of SOD, GSH, and HO-1 were augmented, while the generation of ROS and MDA was diminished. CONCLUSIONS: Our results indicate that COS exhibits therapeutic effects on CAG through the promotion of Nrf2 expression and inhibition of oxidative stress and DNA damage. Therefore, COS has the potential to provide new drugs for the treatment of CAG.

Alleviating sepsis: Revealing the protective role of costunolide in a cecal ligation and puncture rat model.

Objectives: Sepsis poses a significant threat to human life, rendering it a burdensome medical disease. Despite significant advancements, the current state of medical science still lacks a viable and efficacious cure. Costunolide (COST) is a multifaceted sesquiterpene lactone that exhibits a range of actions, including anti-inflammatory and antioxidant properties. We investigated the potential impacts of COST on a rat sepsis model caused by cecal ligation and puncture (CLP). Materials and Methods: We created an experimental rat model with the following groups: SHAM, CLP, CLP+low dose COST, and CLP+high dose COST. Blood, kidney, and lung samples were collected. Inflammatory mediators such as interleukin-1beta (IL-1ß), IL-6, tumor necrosis factor-alpha (TNF- α), and nuclear factor kappa-B (NF-κB) were investigated. In addition, we assessed oxidative stress by measuring 8-Hydroxydeoxyguanosine (8-OHdG) immunopositivity, MDA levels, glutathione (GSH), and superoxide dismutase (SOD) activity. Histopathological and immunohistochemical examinations backed up our findings. Results: Compared to the CLP group, the COST group showed a reduction in inflammatory and oxidative stress indicators. The expression of inflammatory mediators was suppressed by COST, and histological examinations revealed improvements in kidney and lung tissues in the treatment groups. Conclusion: Our study highlights the preventive effects of COST against CLP-induced sepsis-related injury. Considering its beneficial effects against many diseases, COST is worthy as to be evaluated against sepsis.

Sesquiterpene Lactones Containing an α-Methylene-γ-Lactone Moiety Selectively Down-Regulate the Expression of Tumor Necrosis Factor Receptor 1 by Promoting Its Ectodomain Shedding in Human Lung Adenocarcinoma A549 Cells.

Alantolactone is a eudesmane-type sesquiterpene lactone containing an α-methylene-γ-lactone moiety. Previous studies showed that alantolactone inhibits the nuclear factor κB (NF-κB) signaling pathway by targeting the inhibitor of NF-κB (IκB) kinase. However, in the present study, we demonstrated that alantolactone selectively down-regulated the expression of tumor necrosis factor (TNF) receptor 1 (TNF-R1) in human lung adenocarcinoma A549 cells. Alantolactone did not affect the expression of three adaptor proteins recruited to TNF-R1. The down-regulation of TNF-R1 expression by alantolactone was suppressed by an inhibitor of TNF-α-converting enzyme. Alantolactone increased the soluble forms of TNF-R1 that were released into the culture medium as an ectodomain. The structure-activity relationship of eight eudesmane derivatives revealed that an α-methylene-γ-lactone moiety was needed to promote TNF-R1 ectodomain shedding. In addition, parthenolide and costunolide, two sesquiterpene lactones with an α-methylene-γ-lactone moiety, increased the amount of soluble TNF-R1. Therefore, the present results demonstrate that sesquiterpene lactones with an α-methylene-γ-lactone moiety can down-regulate the expression of TNF-R1 by promoting its ectodomain shedding in A549 cells.

Selection and evaluation of quality markers (Q-markers) of vladimiriae radix extract for cholestatic liver injury based on spectrum-effect relationship, pharmacokinetics, and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: As a representative local medicinal herb produced in China, Vladimiriae Radix (VR) has been proven to exert hepatoprotective and choleretic effects, with particular therapeutic efficacy in cholestatic liver injury (CLI), as demonstrated by the VR extract (VRE). However, the quality markers (Q-markers) of VRE for the treatment of CLI remain unclear. AIM OF THE STUDY: A new strategy based on the core element of "efficacy" was proposed, using a combination of spectrum-effect relationship, pharmacokinetics, and molecular docking methods to select and confirm Q-markers of VRE. MATERIAL AND METHODS: First, the HPLC fingerprinting of 10 batches of VRE was studied, and the in vivo pharmacological index of anti-CLI in rats was determined. The spectrum-effect relationship was utilized as a screening method to identify the Q-markers of VRE. Secondly, Q-markers were used as VRE pharmacokinetic markers to measure their concentrations in normal and CLI rat plasma, and to analyze their disposition. Finally, molecular docking was utilized to predict the potential interaction between the identified Q-markers and crucial targets of CLI. RESULTS: The fingerprints of 10 batches of VRE was established. The in vivo pharmacological evaluation of rats showed that VRE had a significant therapeutic effect on CLI. The spectrum-effect correlation analysis showed that costunolide (COS) and dehydrocostus lactone (DEH) were the Q-markers of VRE anti-CLI. The pharmacokinetic results showed that AUC(0-t), Cmax, CLZ/F, and VZ/F of COS and DEH in CLI rats had significant differences (P < 0.01). They were effectively absorbed into the blood plasma of CLI rats, ensuring ideal bioavailability, and confirming their role as Q-markers. Molecular docking results showed that COS, DEH had good affinity with key targets (FXR, CAR, PXR, MAPK, TGR5, NRF2) for CLI treatment (Binding energy < -4.52 kcal mol-1), further verifying the correctness of Q-marker selection. CONCLUSIONS: In this study, through the combination of experimental and theoretical approaches from the aspects of pharmacodynamic expression, in vivo process rules, and interaction force prediction, the therapeutic effect of VRE and Q-markers (COS、DEH) were elucidated. Furthermore, a new idea based on the principle of "efficacy" was successfully proposed for screening and evaluating Q-markers.

Costunolide mitigates inflammation and promotes extracellualr matrix integrity of thoracic aortic dissection by inhibiting NF-κB signaling.

BACKGROUND: Thoracic aortic dissection (TAD) is one of the most fatal cardiovascular diseases. One of its important pathological characteristics is the local inflammatory response. Many studies have found that Macrophage polarization plays an extremely critical role in the inflammatory progression and tissue remodeling of TAD. Costunolide (CTD) has an improving effect on oxidative stress and inflammation in the body. However, whether it can promote the integrity of extracellular matrix in Aortic dissection and its mechanism are still unclear. METHODS: The male C57BL/6J mice were used to construct an animal model of TAD with ß-aminopropionitrile (BAPN) (100 mg/kg/day, lasting for 28 days), and then CTD (10 mg/kg or 100 mg/kg) was injected intraperitoneally for 28 days to check the survival rate, TAD incidence, aortic morphology and other indicators of the mice. Using hematoxylin-eosin (HE), Masson, Elastin van Gieson (EVG) staining, immunofluorescence (IF), and immunohistochemical staining, the study aimed to determine the therapeutic effects of CTD on an animal model with BAPN-induced TAD. To enhance the examination of the regulatory mechanism of CTD, we conducted transcriptome sequencing on arterial tissues of mice in both the BAPN group and the BAPN + CTD100 group. Next, ANG II were used to construct TAD model in vascular smooth muscle cells (VMSCs). The effects of CTD on the proliferation, migration, invasion, and apoptosis of ANG II-induced cells are to be detected. The expression of MMP2, MMP9, P65, and p-P65 in each group will be examined using Western blot. Finally, the overexpression of IκB kinaseß (IKKß) will be established in VMSCs cells to further explore the protective function of CTD. RESULTS: The result showed that CTD significantly inhibited BAPN induced mortality and TAD incidence in the animal model, improved aortic vascular morphology, promoted the integrity of extracellular matrix in TAD, reduced tissue inflammation, reduced the accumulation of M1 macrophage, promoted M2 macrophage polarization, and reduced the expression of NF-κB pathway related proteins. Mechanistically, CTD significantly weakened the proliferation, migration, invasion, and apoptosis. p-P65 protein expression of TAD cells were induced by ANG II and IKK-ß. CONCLUSION: CTD has the potential to alleviate inflammation, VSMC apoptosis, MMP2/9 levels, and enhance extracellular matrix integrity in TAD by inhibiting the NF-κB signaling pathway.

Costunolide attenuates high-fat diet-induced inflammation and oxidative stress in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a progressive disease that can further evolve towards liver fibrosis and hepatocellular carcinoma in the end stage. Costunolide (Cos) is a natural sesquiterpene lactone that exhibits both anti-inflammatory and antioxidant properties. However, the therapeutic effect of Cos on NAFLD is not clear. In this study, we explored the potential protective effect and mechanism of Cos on NAFLD. C57BL/6 mice were fed with high-fat diet (HFD) to induce NAFLD. Cos was administered by gavage to observe the effect of Cos on NAFLD. We demonstrated that oral administration of Cos reduced HFD-induced hepatic fibrosis and the release of inflammatory cytokines, limiting the generation of reactive oxygen species. In vitro experiments revealed that pretreatment with Cos significantly decreased PA-induced production of inflammatory cytokines and fibrosis in AML-12 cells. Mechanism study showed that the effect of Cos was correlated to the induction of Nrf-2 and inhibition of NF-κB pathways. Collectively, these findings indicated that Cos exerts hepatoprotective effect against NAFLD through blocking inflammation and oxidative stress. Our study suggested that Cos might be an effective pharmacotherapy for the treatment of NAFLD.

A Novel Approach: Targeting Virulence Factors of Candida albicans with Essential Oil Components.

AIMS: Focusing on phytochemicals to target the virulence factors of Candida albicans is a promising avenue for novel antifungal compounds. Given the limited prior research on essential oil (EO) components and their specific effects on C. albicans virulence, our study aimed to explore their impact and uncover the underlying mechanisms. METHODS AND RESULTS: We examined the effects on viability, dimorphic transition, biofilm formation, and changes in the expression of critical virulence-related genes. The results showed that Dehydrocostus Lactone, displayed the most potent growth-inhibiting activity with the lowest MIC value, followed by Thymol, and Costunolide. A substantial, dose-dependent decrease in germ tube formation occurred after exposure to sub-inhibitory concentrations of the EO components, with Carvacrol, Dehydrocostus Lactone, and Thymol exerting the most potent inhibitory effects. Across sub-inhibitory concentrations, Alpha Bisabolol consistently showcased the most potent antibiofilm activity followed by lower but significant inhibitory effects with Dehydrocostus Lactone, Thymol, Alpha Pinene, Costunolide, Carvone, and Carvacrol. Alpha Bisabolol, Alpha Pinene and Dehydrocostus Lactone caused almost total downregulation of ACT1 whilst minimal changes occurred in expression of HWP1, SAP4, ALS3 and ECE1. CONCLUSIONS: Considering that actin is essential for various cellular processes, including budding, cell shape maintenance, and the formation of filaments in C. albicans, it is a plausible hypothesis that inhibiting ACT1 or disturbing actin's normal functioning could potentially affect the fungus's virulence, which warrants additional research and exploration. This study underscores the potent antifungal and anti-virulence properties of various EO components which effectively cripple C. albicans and reduce its disease-causing ability. This innovative approach holds promise for effective clinical therapies.

Costunolide attenuates LPS-induced inflammation and lung injury through inhibiting IKK/NF-κB signaling.

Inflammation is an important pathological process of many acute and chronic diseases, such as sepsis, arthritis, and cancer. Many factors can lead to an inflammatory state of the body, among which bacterial infection plays an important role. Bacterial infection often leads to sepsis, acute lung injury (ALI), or its more serious form of acute respiratory distress syndrome, which are the main fatal diseases in intensive care units. Costunolide has been reported to possess excellent anti-inflammatory activity; however, whether it can affect inflammation induced by gram-negative bacterial is still unclear. Lipopolysaccharide (LPS) stimulated mouse peritoneal macrophages (MPMs) to release proinflammatory cytokines was used as the cell model. The mouse model of sepsis and ALI was built through injecting intravenously and intratracheally of LPS. In the present study, costunolide inhibited LPS-induced inflammatory response through IKK/NF-κB signaling pathway in macrophages. In vivo, costunolide attenuated LPS-induced septic death in mice. Meanwhile, costunolide treatment alleviated LPS-induced lung injury and inflammation via inhibiting the infiltration of inflammatory cells and the expression of inflammatory cytokines. Taken together, these results demonstrated that costunolide could attenuate gram-negative bacterial induced inflammation and diseases and might be a potential candidate for the treatment of inflammatory diseases.

Exploration of costunolide derivatives as potential anti-inflammatory agents for topical treatment of atopic dermatitis by inhibiting MAPK/NF-κB pathways.

Atopic dermatitis (AD) is a common inflammatory disease and it is very difficult to treat. In the present work, a series of costunolide derivatives have been prepared, and in vitro and in vivo anti-inflammatory activities have evaluated. The results showed that most derivatives displayed good inhibition of NO generation with low cytotoxicity, and 7d could inhibit the phosphorylation of P38, P65 NF-κB and IκB-α in LPS-induced RAW264.7 model. The in vivo researches showed that 7d could improve skin injury symptoms, decrease Th2-type cytokine levels, inhibit HIS levels, alleviate scratching and repaire the damaged skin barrier through the inhibition of phosphorylation of MAPK and NF-κB signaling pathways on MC903-induced AD model. Therefore, costunolide derivatives may be new potent anti-AD agents for further study.

Hepatoprotective effect of syringin combined with costunolide against LPS-induced acute liver injury in L-02 cells via Rac1/AKT/NF-κB signaling pathway.

Acute liver injury (ALI) leads to abnormal liver function and damage to liver cells. Syringin (syr) and costunolide (cos) are the major extracts from Dolomiaea souliei (Franch.) C.Shih (D. souliei), showing diverse biological functions in various biological processes. We explored the underlying hepatoprotective effects of syr+cos against LPS-induced ALI. Cell viability and proliferation were assessed using an MTT assay and immunofluorescence staining. Flow cytometry analysis was used to detect cell cycle distribution and apoptosis. ELISA was utilized to measure liver function and antioxidant stress indexes. qRT-PCR and western blotting was performed to determine mRNA and protein levels respectively. Using shRNA approach to Rac1 analyzed transcriptional targets. The results showed that syr+cos promoted L-02 cell proliferation, inhibiting the cell apoptosis and blocking cell cycle in G1 and G2/M phase. Syr+cos decreased the production of ALT, AST, LDH, MDA and ROS while increased SOD and CAT activities. Pretreated with syr+cos may decrease expressions of caspase-3,7,9, NF-κB, TNF-α proteins, Cyclin B, CDK1 and p-IκB proteins while p-IκB increased. Silencing of Rac-1 may protect the liver by increasing AKT, S473, T308 and reducing p-AKT proteins. Syr+cos exhibits anti-ALI activity via Rac1/AKT/NF-κB signaling pathway which might act as an effective candidate drug for the treatment of ALI.

Effects of Sesquiterpene Lactones on Primary Cilia Formation (Ciliogenesis).

Sesquiterpene lactones (SLs), plant-derived metabolites with broad spectra of biological effects, including anti-tumor and anti-inflammatory, hold promise for drug development. Primary cilia, organelles extending from cell surfaces, are crucial for sensing and transducing extracellular signals essential for cell differentiation and proliferation. Their life cycle is linked to the cell cycle, as cilia assemble in non-dividing cells of G0/G1 phases and disassemble before entering mitosis. Abnormalities in both primary cilia (non-motile cilia) and motile cilia structure or function are associated with developmental disorders (ciliopathies), heart disease, and cancer. However, the impact of SLs on primary cilia remains unknown. This study evaluated the effects of selected SLs (grosheimin, costunolide, and three cyclocostunolides) on primary cilia biogenesis and stability in human retinal pigment epithelial (RPE) cells. Confocal fluorescence microscopy was employed to analyze the effects on primary cilia formation (ciliogenesis), primary cilia length, and stability. The effects on cell proliferation were evaluated by flow cytometry. All SLs disrupted primary cilia formation in the early stages of ciliogenesis, irrespective of starvation conditions or cytochalasin-D treatment, with no effect on cilia length or cell cycle progression. Interestingly, grosheimin stabilized and promoted primary cilia formation under cilia homeostasis and elongation treatment conditions. Thus, SLs have potential as novel drugs for ciliopathies and tumor treatment.

Optimized bilosome-based nanoparticles enhance cytotoxic and pro-apoptotic activity of costunolide in LS174T colon cancer cells.

Costunolide (COST) is a sesquiterpene lactone that belongs to the germacranolide group, and occurs mainly in Saussurea lappa Clarke. Although COST inhibits the proliferation and metastasis of cancer cells and induces their apoptosis, it suffers poor water solubility and cellular permeability. Therefore, this study aimed to enhance the anti-proliferative activity of COST in LS174T colon cancer cells through its inclusion in bilosomal nanoformulation (COST-BILs). The optimized BIL formula contained cholesterol and Span-85 in a molar ratio of 1:5 as well as bile salt at a molar concentration of 0.5 mM, with entrapment efficiency of 63.4 ± 3.59 % and particle size of 119.7 ± 3.63 nm. The optimized COST-BILs showed a potent cytotoxic effect against LS174T cells with an IC50 of 6.20 µM; meanwhile, raw COST had an IC50 of 15.78 µM. Safety and relative selectivity were confirmed in the normal human colonic epithelial cells (HCoEpC). Cell cycle analysis indicated that both raw COST and COST-BILs significantly increased the fraction of LS174T cells in the sub-G1 phase. This was accompanied by a significant enhancement of early, late, and total apoptosis, as indicated by annexin-V staining. In addition, COST-BILs exhibited more potent activity in up-regulating CASP3, TP53, and BAX, and in down-regulating the expression of BCL2 mRNA as compared to raw COST. Further, the prepared formula enhanced the release of cytochrome C as well as the generation of reactive oxygen species (ROS) and reduced the integrity of mitochondrial membranes. In conclusion, the loading of COST on BILs significantly enhances its pro-apoptotic activity in LS174T cells.

Three sesquiterpene lactones suppress lung adenocarcinoma by blocking TMEM16A-mediated Ca2+-activated Cl- channels.

Transmembrane protein TMEM16A, which encodes calcium-activated chloride channel has been implicated in tumorigenesis. Overexpression of TMEM16A is associated with poor prognosis and low overall survival in multiple cancers including lung adenocarcinoma, making it a promising biomarker and therapeutic target. In this study, three structure-related sesquiterpene lactones (mecheliolide, costunolide and dehydrocostus lactone) were extracted from the traditional Chinese medicine Aucklandiae Radix and identified as novel TMEM16A inhibitors with comparable inhibitory effects. Their effects on the proliferation and migration of lung adenocarcinoma cells were examined. Whole-cell patch clamp experiments showed that these sesquiterpene lactones potently inhibited recombinant TMEM16A currents in a concentration-dependent manner. The half-maximal concentration (IC50) values for three tested sesquiterpene lactones were 29.9 ± 1.1 µM, 19.7 ± 0.4 µM, and 24.5 ± 2.1 µM, while the maximal effect (Emax) values were 100.0% ± 2.8%, 85.8% ± 0.9%, and 88.3% ± 4.6%, respectively. These sesquiterpene lactones also significantly inhibited the endogenous TMEM16A currents and proliferation, and migration of LA795 lung cancer cells. These results demonstrate that mecheliolide, costunolide and dehydrocostus lactone are novel TMEM16A inhibitors and potential candidates for lung adenocarcinoma therapy.

Costunolide and parthenolide: Novel blood-brain barrier permeable sesquiterpene lactones to improve barrier tightness.

Sesquiterpene lactones - such as those found in chicory - are considered promising bioactive compounds. These small molecules have shown several health benefits for various diseases, including brain disorders. However, it is unknown whether these compounds can cross the blood-brain barrier (BBB), and which could be the effects on brain microvascular endothelial cells. We show that six sesquiterpene lactones evaluated in an in vitro model of the BBB have different capacities to be transported through the barrier. Costunolide presented more than 20 % of transport while lactucin, 11ß-13-dihydrolactucin, 11ß-13-dihydrolactucopicrin, and parthenolide presented between 10 % and 20 %, whilst almost no transport was detected for lactucopicrin. Furthermore, costunolide and parthenolide reduced P-gp ABC transporter expression alongside an increase in caveolin-1, the main protein of caveolae. Remarkably, these two compounds improved barrier tightness by increasing the expression of both tight and adherens junctions. These findings open a new avenue to explore costunolide and parthenolide as promising compounds for brain therapies.

Costunolide prevents renal ischemia-reperfusion injury in rats by reducing autophagy, apoptosis, inflammation, and DNA damage.

Objectives: Renal ischemia-reperfusion (I/R) is a vital health condition leading to acute kidney injury. Costunolide (COST) is an actively used molecule clinically for its anti-inflammatory, antioxidant, and immunomodulatory properties. In the present study, we searched for the possible protective effects of COST against renal ischemia/reperfusion (I/R) injury in rats. Materials and Methods: We established a renal I/R rat model. We divided forty rats into four groups: group I (sham), group II (I/R), group III (I/R+COST 5 mg/kg), and group IV (I/R+COST 10 mg/kg). We collected blood, kidney, and lung samples for analysis. Results: COST administration performed anti-oxidant and anti-inflammatory activity by reducing oxidant parameters and proinflammatory cytokine levels. COST alleviated DNA damage through declining 8-hydroxydeoxyguanosine (8-OHdG) levels. In addition, COST diminished tubular damage and inflammation by reducing kidney injury molecule-1 (KIM-1) production. COST administration also ameliorated apoptosis and autophagy by decreasing caspase-3 and microtubule-associated protein light chain 3B (MAPLC3, LC3B) expression. Conclusion: COST demonstrated protective effects against renal I/R-induced injury.

Costunolide alleviated DDC induced ductular reaction and inflammatory response in murine model of cholestatic liver disease.

Purpose: Cholestatic liver diseases are groups of hepatobiliary diseases without curative drug-based therapy options. Regulation of bile acid (BA) metabolism, hepatoperiductal fibrosis, and inflammatory response indicated present novel methods for the treatment of cholestatic liver disease. Costunolide (COS) from herb Saussurea lappa exerts a pharmacological effect of regulation of BA metabolism, liver fbrosis and inflammatory response. The present study aimed to clarify the pharmacodynamic effects of COS against the murine model of cholestatic liver disease. Methods: We established a murine model of cholestatic liver disease through chronic feeding of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 28 days. Two independent in vivo experiments were designed to reveal the pharmacological effect of COS against cholestatic liver disease. In the first experiment, two dosages of COS (10 and 30 mg/kg) were intraperitoneally injected into model mice daily for 14 days. In the second experiment, high dosage of COS (30 mg/kg) was intraperitoneally injected into control and model mice daily for 28 days. Results: In the evaluation of the hepatoprotective effect of COS, COS showed dosage-dependent improvement of cholestatic liver disease, including ductular reaction, hepatoperiductal fibrosis, and inflammatory response. The mechanism of COS-mediated hepatoprotective effects mainly relies on the regulation of BA metabolism, and the inflammatory response. DDC diet feed induced hepatic BA metabolism, transport and circulation dysfunction. COS treatment not only regulated the BA metabolism and transport gene, but also reprogrammed hepatic primary and secondary BA concentrations. DDC induced hepatic infiltrated monocytes derived macrophages and lymphocytes were inhibited, while Kupffer cells were preserved by COS treatment. The liver elevating inflammatory cytokines of DDC diet feed were alleviated by COS. Moreover, high dosage of 30 mg/kg COS treatment for 28 days resulted in no significant serological changes and no obvious hepatic histopathological changes when compared with control mice. Conclusion: COS protected against DDC diet feeding-induced cholestatic liver disease since COS regulated BA metabolism, ductular reaction, hepatoperiductal fibrosis and inflammatory response. COS is suggested as a potential natural product for the treatment of cholestatic liver disease.

HPLC-PDA Method for Quantification of Bioactive Compounds in Crude Extract and Fractions of Aucklandia costus Falc. and Cytotoxicity Studies against Cancer Cells.

Aucklandia costus Falc. (Synonym: Saussurea costus (Falc.) Lipsch.) is a perennial herb of the family Asteraceae. The dried rhizome is an essential herb in the traditional systems of medicine in India, China and Tibet. The important pharmacological activities reported for Aucklandia costus are anticancer, hepatoprotective, antiulcer, antimicrobial, antiparasitic, antioxidant, anti-inflammatory and anti-fatigue activities. The objective of this study was the isolation and quantification of four marker compounds in the crude extract and different fractions of A. costus and the evaluation of the anticancer activity of the crude extract and its different fractions. The four marker compounds isolated from A. costus include dehydrocostus lactone, costunolide, syringin and 5-hydroxymethyl-2-furaldehyde. These four compounds were used as standard compounds for quantification. The chromatographic data showed good resolution and excellent linearity (r2 ˃ 0.993). The validation parameters, such as inter- and intraday precision (RSD < 1.96%) and analyte recovery (97.52-110.20%; RSD < 2.00%),revealed the high sensitivity and reliability of the developed HPLC method. The compounds dehydrocostus lactone and costunolide were concentrated in the hexane fraction (222.08 and 65.07 µg/mg, respectively) and chloroform fraction (99.02 and 30.21 µg/mg, respectively), while the n-butanol fraction is a rich source of syringin (37.91 µg/mg) and 5-hydroxymethyl-2-furaldehyde (7.94 µg/mg). Further, the SRB assay was performed for the evaluation of anticancer activity using lung, colon, breast and prostate cancer cell lines. The hexane and chloroform fractions show excellent IC50 values of 3.37 ± 0.14 and 7.527 ± 0.18 µg/mL, respectively, against the prostate cancer cell line (PC-3).

Therapeutic Effect of Costunolide in Autoimmune Hepatitis: Network Pharmacology and Experimental Validation.

Novel treatments for autoimmune hepatitis (AIH) are highly demanded due to the limitations of existing therapeutic agents. Costunolide is a promising candidate due to its anti-inflammatory and hepatoprotective function, but its effect in AIH remains obscure. In this study, we integrated network pharmacology and experimental validation to reveal the effect and mechanism of costunolide in AIH. A total of 73 common targets of costunolide and AIH were obtained from databases. Pathway enrichment analysis indicated that PI3K-AKT pathway was the core pathway of costunolide in AIH. Protein-protein interaction network analysis and molecular docking revealed that SRC and IGF1R might play critical roles. In two murine AIH models, costunolide significantly attenuated liver injury, inflammation, and fibrosis reflected by the liver gross appearance, serum transaminases, necrosis area, spleen index, immune cell infiltration, and collagen deposition. Western blot and immunohistochemistry confirmed that phosphorylated AKT, SRC, and IGF1R were upregulated in AIH models, and costunolide administration could inhibit the phosphorylation of these proteins. In summary, costunolide significantly ameliorates murine AIH. The therapeutic effect might work by suppressing the activation of PI3K-AKT pathway and inhibiting the phosphorylation of SRC and IGF1R. Our research reveals the potent therapeutic effect of costunolide in AIH and the potential role of SRC and IGF1R in AIH for the first time, which may further contribute to the novel drug development for AIH and other autoimmune diseases.

Costunolide covalently targets and inhibits CaMKII phosphorylation to reduce ischemia-associated brain damage.

BACKGROUND: Chronic cerebral hypoperfusion (CCH) is a leading cause of disability and mortality worldwide. Restoring cerebral blood flow (CBF) through vasodilatation is particularly important in the treatment of CCH. Costunolide (Cos) is a natural sesquiterpenoid compound with vasodilatory effect, but its mechanism is unclear. PURPOSE: This study aimed to investigate the vasodilatory mechanism of Cos and provide a new therapeutic regimen for treating CCH. METHODS: The therapeutic effect of Cos on CCH was assessed in a rat model of permanent common carotid artery occlusion. The direct target protein for improving CBF was identified by drug affinity responsive target stability combined with quantitative differential proteomics analysis. The molecular mechanism of Cos acting on its target protein was analyzed by multidisciplinary approaches. The signalling involved was assessed using site-directed pharmacological intervention. RESULTS: Cos has a significant therapeutic effect on ischemic brain injury by restoring CBF. Multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) was identified as a direct target of the natural small molecule Cos with a therapeutic effect on CCH. Mechanistic studies revealed that the α,ß-unsaturated-γ-lactone ring of Cos covalently binds to the Cys116 residue of CaMKII. It then inhibits the phosphorylation of CaMKII and reduces the calcium concentration in vascular smooth muscle cells, thus playing a role in vasodilation and increasing CBF. Notably, this covalent binding between Cos and CaMKII can exert a long-term vasodilator activity. CONCLUSION: We reported for the first time that Cos reduced ischemia-associated brain damage by covalently binding to the Cys116 residue of CaMKII, inhibiting CaMKII phosphorylation, and exerting long-term vasodilatory activity. This study not only found a new covalent inhibitor against the phosphorylation of CaMKII but also suggested that pharmacologically targeting CaMKII is a promising therapeutic strategy for CCH.