Certain plant species within the Apiales order accumulate triterpenoid saponins featuring a distinctive glucose-glucose-rhamnose (G-G-R) sugar chain attached at the C-28 position of the pentacyclic triterpene skeleton. Until recently, the genomic basis underlying the biosynthesis and evolution of the sugar chain remains elusive. In this study, we identified two novel glycoside glycosyltransferases (GGTs) that could sequentially install the sugar chain\'s second D-glucose and the third L-rhamnose during the biosynthesis of asiaticoside and madecassoside, two representative G-G-R sugar chain-containing triterpenoid saponins produced by Centella asiatica. Enzymatic assays unveiled the remarkable substrate promiscuity of the two GGTs and key residues crucial for the sugar-donor selectivity of the glucosyltransferase and rhamnosyltransferase. We further revealed syntenic tandem gene duplicates of the two GGTs in the Apiaceae and Araliaceae families, suggesting a well-conserved genomic basis underlying the sugar chain assembly that likely evolved in the early ancestors of the Apiales order. Moreover, the expression pattern of the two GGTs in pierced leaves of C. asiatica was found to be correlated with the production of asiaticoside and madecassoside, implying their involvement in the host\'s defense against herbivores and pathogens. Our work sheds light on the biosynthesis and evolution of complex saponin sugars, paving the way for future engineering of diverse bioactive triterpenoids with unique glycoforms.

Non-small cell lung cancer (NSCLC) is the primary inducer of cancer-related death worldwide. Asiaticoside (ATS) is a triterpenoid saponin that has been indicated to possess an antitumor activity in several malignancies. Nonetheless, its detailed functions in NSCLC remain unclarified. In this study, NSCLC cells were exposed to various doses of ATS. Functional experiments were employed to estimate the ATS effect on NSCLC cell behaviors. Western blotting was implemented for protein expression evaluation. A xenograft mouse model was established to assess the ATS effect on NSCLC in vivo. The results showed that ATS restrained NSCLC cell proliferation, cell cycle progression, migration, and invasiveness. ATS reversed TGF-β-induced promotion in epithelial-mesenchymal transition (EMT). Mechanistically, ATS inhibited Wnt/β-catenin signaling in NSCLC. Upregulating β-catenin restored ATS-mediated suppression of NSCLC cell aggressiveness. Moreover, ATS administration repressed tumorigenesis in tumor-bearing mice. In conclusion, ATS represses growth and metastasis in NSCLC by blocking EMT via the inhibition of Wnt/β-catenin signaling.

Rivastigmine hydrogen tartrate (RHT), a reversible cholinesterase inhibitor, is considered as the first-line therapy for mild to moderate Alzheimer\'s disease. Asiaticoside (AS), a pentacyclic triterpenoid saponin, is well known as cognitive enhancer due to its antioxidant effect. Based on the hypothesis of their synergistic therapeutic potential, RHT and AS were co-encapsulated in niosomal formulation. A simple, precise, and accurate high-performance liquid chromatography method was developed for simultaneous quantitative analysis. The chromatographic parameters were optimized by Box-Behnken experimental design. The separation was performed on a reversed-phase Phenomenex C18 (150 mm × 4.6 mm, 5 μm) column at 30 °C under the UV detection of 210 nm. The optimized mobile phase consisted of a mixture of 20 mM potassium dihydrogen phosphate buffer (pH 2.6) and acetonitrile (72:28 % v/v) under the isocratic mode at the flow rate of 0.9 mL/min. The developed method was fully validated under the ICH guidelines and could be successfully applied for simultaneous quantitative analysis of RHT and AS in niosomal formulation.

UNASSIGNED: Asiaticoside (AS) has been reported to improve the changes induced by high glucose stimulation, and it may have potential therapeutic effects on gestational diabetes mellitus (GDM). This study aims to explore the effect of AS on the cell model of GDM and the action mechanism of the PI3K/AKT pathway.
UNASSIGNED: The GDM model was established in HTR-8/Svneo cells with a high glucose (HG) medium. After the cytotoxicity assay of AS, cells were divided into the control group, HG group and HG + AS group to conduct control experiment in cells. The cell proliferation and migration were detected by CCK-8 assay and scratch test, respectively. The mRNA levels of PI3K, AKT2, mTORC1, and GLUT4 in PI3K/AKT signalling pathway were measured by RT-PCR, and the protein expressions of these signalling molecules were monitored by western blot.
UNASSIGNED: AS showed a promotion effect on the cell proliferation rate of HTR-8/Svneo cells, and 80 μmol/L AS with a treatment time of 48 h had no cytotoxicity. The cell proliferation rate, migration rate, mRNA levels and protein expressions of PI3K, AKT2, mTORC1, and GLUT4 in the HG group were significantly lower than those in the control group, which were significantly increased in the HG + AS group (p < 0.05).
UNASSIGNED: AS can facilitate the cell proliferation and migration in the cell model of GDM, and might play a role in GDM treatment via PI3K/AKT pathway.
Asiaticoside possesses various pharmacological effects and has been reported to show a beneficial effect on the treatment of diabetes mellitus. This research firstly investigated the effect and mechanism of asiaticoside on gestational diabetes mellitus, and found that asiaticoside could facilitate the cell proliferation and migration of HTR-8/Svneo cells treated with high glucose, and affect the signalling molecules of PI3K/AKT pathway. Therefore, asiaticoside may be a novel useful therapeutic drug in the treatment of gestational diabetes mellitus.

BACKGROUND: Our previous study has revealed that asiaticoside (AC) promotes endoplasmic reticulum stress and antagonizes proliferation and migration of gastric cancer (GC) via miR-635/HMGA1 axis. However, the effect and mechanism of AC on other progressions of GC, such as ferroptosis and immune escape, are still unknown.
METHODS: AGS and HGC27 cells were incubated with 1, 2 and 4 μM of AC for 24 h. Mice xenografted with AGS cells were intragastrically injected with AC. The effect and mechanism of AC on GC were determined by the measurement of the ferrous iron level, the ROS level and the glutathione peroxidase (GSH) content, flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and western blotting assays.
RESULTS: AC increased the Fe2+ level and the ROS level, but decreased the expression of GPX4 and SLC7A11 and the GSH level. Besides, AC enhanced the percent of CD8+ T cells and the IFN-γ concentration, but reduced the PD-L1 expression and the IL-10 level. Mechanically, AC downregulated the relative levels of β-catenin, active-β-catenin, p-GSK3β/GSK3β, cyclin D1 and c-Myc in GC cells, which were rescued with the application of LiCl (an activator of Wnt/β-catenin pathway) in AGS cells. Moreover, activation of Wnt/β-catenin pathway by LiCl or the β-catenin overexpression inverted the effect of AC on ferroptosis and immune escape in GC cells. In vivo, AC treatment declined the tumor size and weight, the level of GPX4, SLC7A11, PD-L1 and IFN-γ, and the expression of Wnt/β-catenin pathway.
CONCLUSIONS: AC enhanced ferroptosis and repressed immune escape by downregulating the Wnt/β-catenin signaling in GC.

Acute lung injury (ALI) is a common disease with complex pathogenesis. However, the treatment is mainly symptomatic with limited clinical options. Asiaticoside (AS), a Chinese herbal extract, has protective effects against LPS-induced ALI in mice and inhibits nitric oxide and prostaglandin E2 synthesis; however, the specific mechanism of AS in the prevention and treatment of LPS-induced ALI needs further study. Sema4D/CD72 pathway, mitochondrial dysfunction, and miRNA-21 are closely associated with inflammation. Therefore, the present study aimed to explore whether AS exerts its therapeutic effect on ALI by influencing Sema4D/CD72 pathway and mitochondrial dysfunction, restoring the balance of inflammatory factors, and influencing miRNA-21 expression. Cell and animal experiments were performed to investigate the effect of AS on ALI. Lipopolysaccharide (LPS) was used to establish the ALI model. CCK8 and flow cytometry were used to detect the cell viability and apoptosis rate. HE staining and wet-to-dry weight ratio (W/D) of lung tissue were determined. The expressions of Sema4D, CD72, NF-κB p65, Bax, Bcl2, and caspase 3 in RAW264.7 cells and lung tissues were detected by western blot, and the levels of IL-10 and IL-1β induced by LPS in supernatant of RAW264.7 cells and BALF were measured by ELISA. And the expression of miRNA-21 in cells and lung tissues was detected by fluorescence quantitative PCR. The result shows that AS treatment suppressed LPS-induced cell damage and lung injury in mice. AS treatment could alleviate the pathological changes such as inflammatory infiltration and histopathological changes in the lungs caused by LPS, and reduce the ratio of W/D. AS significantly alleviated the decrease of mitochondrial membrane potential induced by LPS, inhibited the increase of ROS production, and reduced the expression of mitochondrial fission proteins Drp1 and Fis1. The high-dose AS group significantly downregulated the expression of Sema4D, CD72, phosphorylated NF-κB p65, and apoptosis-related proteins, decreased the pro-inflammatory factor IL-1β, and enhanced the level of anti-inflammatory factor IL-10. In addition, AS promoted miRNA-21 expression. These effects inhibited apoptosis and restored the balance between anti- and pro-inflammatory factors. This represents the inaugural report elucidating the mechanism by which AS inhibits the Sema4D/CD72 signaling pathway. These findings offer novel insights into the potential application of AS in both preventing and treating ALI.

OBJECTIVE: To investigate the effect of asiaticoside on blood pressure and relaxation of thoracic aorta in rats and explore the underlying mechanism.
METHODS: SD rats treated with 50 and 100 mg/kg asiaticoside by daily gavage for 2 weeks were monitored for systolic blood pressure changes, and histological changes of the thoracic aorta were evaluated using HE staining. In isolated rat endothelium-intact and endothelium-denuded thoracic aorta rings, the effects of asiaticoside on relaxation of the aortic rings were tested at baseline and following norepinephrine (NE)- and KCl-induced constriction. The vascular relaxation effect of asiaticoside was further observed in NE-stimulated endothelium-intact rat aortic rings pretreated with L-nitroarginine methyl ester, indomethacin, zinc protoporphyrin Ⅸ, tetraethyl ammonium chloride, glibenclamide, barium chloride, Iberiotoxin, 4-aminopyridine, or TASK-1-IN-1. The aortic rings were treated with KCl and NE followed by increasing concentrations of CaCl2 to investigate the effect of asiaticoside on vasoconstriction induced by external calcium influx and internal calcium release.
RESULTS: Asiaticoside at 50 and 100 mg/kg significantly lowered systolic blood pressure in rats without affecting the thoracic aorta histomorphology. While not obviously affecting resting aortic rings with intact endothelium, asiaticoside at 100 mg/kg induced significant relaxation of the rings constricted by KCl and NE, but its effects differed between endothelium-intact and endothelium-denuded rings. In endothelium-intact aortic rings pretreated with indomethacin, ZnPP Ⅸ, barium chloride, glyburide, TASK-1-IN-1 and 4-aminopyridine, asiaticoside did not produce significant effect on NE-induced vasoconstriction, and tetraethylammonium, Iberiotoxin and L-nitroarginine methyl ester all inhibited the relaxation effect of asiaticoside. In KCland NE-treated rings, asiaticoside obviously inhibited CaCl2-induced vascular contraction.
CONCLUSIONS: Asiaticoside induces thoracic aorta relaxation by mediating high-conductance calcium-activated potassium channel opening, promoting nitric oxide release from endothelial cells and regulating Ca2+ influx and outflow, thereby reducing systolic blood pressure in rats.

OBJECTIVE: This study aims to evaluate the impact of asiaticoside (AC) on the viability and proliferation of dental pulp stem cells (DPSCs), considering the known negative effects of routinely used intracanal medicaments. This evaluation will be compared with the outcomes from using traditional intracanal medicaments, specifically triple antibiotic paste (TAP) and calcium hydroxide [Ca(OH)2].
METHODS: The DPSCs were obtained from the third molars of an adult donor. The application of flow cytometry was employed to do a phenotypic analysis on DPSCs using CD90, CD73, CD105, CD34, CD14, and CD45 antibodies. The methylthiazol tetrazolium (MTT) assay was employed to assess cellular viability. The cells were treated with different concentrations of TAP and Ca(OH)2 (5, 2.5, 1, 0.5, and 0.25 mg/mL), along with AC (100, 50, 25, 12.5, and 6.25 µM). A cell proliferation rate was performed at 3, 5, and 7 days.
RESULTS: The characterization of DPSCs was conducted by flow cytometry analysis, which verified the presence of mesenchymal cell surface antigen molecules (CD105, CD73, and CD90) and demonstrated the absence of hematopoietic markers (CD34, CD45, and CD14). Cells treated with concentrations over 0.5 mg/mL of TAP and Ca(OH)2 showed a notable reduction in cell viability in comparison to the untreated cells (p < 0.05). Additionally, the cells treated with different concentrations of AC 12.5, 6.25, 25, and 50 µM did not differ significantly from the untreated cells (p > 0.05). Nevertheless, cells treated with concentrations of 100 µM showed a significant reduction in viability compared to the untreated cells (p < 0.05). After a period of 7 days, it was noted that cells exposed to three different concentrations of AC (50, 25, and 12.5 µM) had a notable rise in cell density in comparison to TAP and Ca(OH)2 (p < 0.05). Furthermore, cells that were exposed to a concentration of 12.5 µM exhibited the highest cell density.
CONCLUSIONS: The cellular viability of the AC-treated cells was superior to that of the TAP and Ca(OH)2-treated cells. Moreover, the AC with a concentration of 12.5 µM had the highest degree of proliferation.
CONCLUSIONS: This study underscores the importance of evaluating alternative root canal medicaments and their effects on DPSCs\' growth and vitality. The findings on AC, particularly its influence on the survival and proliferation of DPSCs, offer valuable insights for its probable use as an intracanal medication. This research contributes to the ongoing efforts to identify safer and more effective intracanal treatments, which are crucial for enhancing patient outcomes in endodontic procedures. How to cite this article: Alazemi MJ, Badawi MF, Elbeltagy MG, et al. Examining the Effects of Asiaticoside on Dental Pulp Stem Cell Viability and Proliferation: A Promising Approach to Root Canal Treatment. J Contemp Dent Pract 2024;25(2):118-127.

BACKGROUND: Parkinson\'s disease (PD), a neurodegenerative disorder, is characterized by motor symptoms due to the progressive loss of dopaminergic neurons in the substantia nigra (SN) and striatum (STR), alongside neuroinflammation. Asiaticoside (AS), a primary active component with anti-inflammatory and neuroprotective properties, is derived from Centella asiatica. However, the precise mechanisms through which AS influences PD associated with inflammation are not yet fully understood.
OBJECTIVE: This study aimed to explore the protective mechanism of AS in PD.
METHODS: Targets associated with AS and PD were identified from the Swiss Target Prediction, Similarity Ensemble Approach, PharmMapper, and GeneCards database. A protein-protein interaction (PPI) network was constructed to identify potential therapeutic targets. Concurrently, GO and KEGG analyses were performed to predict potential signaling pathways. To validate these mechanisms, the effects of AS on 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD in mice were investigated. Furthermore, neuroinflammation and the activation of the NLRP3 inflammasome were assessed to confirm the anti-inflammatory properties of AS. In vitro experiments in BV2 cells were then performed to investigate the mechanisms of AS in PD. Moreover, CETSA, molecular docking, and molecular dynamics simulations (MDs) were performed for further validation.
RESULTS: Network pharmacology analysis identified 17 potential targets affected by AS in PD. GO and KEGG analyses suggested the biological roles of these targets, demonstrating that AS interacts with 149 pathways in PD. Notably, the NOD-like receptor signaling pathway was identified as a key pathway mediating AS\'s effect on PD. In vivo studies demonstrated that AS alleviated motor dysfunction and reduced the loss of dopaminergic neurons in MPTP-induced PD mice. In vitro experiments demonstrated that AS substantially decreased IL-1β release in BV2 cells, attributing this to the modulation of the NLRP3 signaling pathway. CETSA and molecular docking studies indicated that AS forms a stable complex with NLRP3. MDs suggested that ARG578 played an important role in the formation of the complex.
CONCLUSIONS: In this study, we first predicted that the potential target and pathway of AS\'s effect on PD could be NLRP3 protein and NOD-like receptor signaling pathway by network pharmacology analysis. Further, we demonstrated that AS could alleviate symptoms of PD induced by MPTP through its interaction with the NLRP3 protein for the first time by in vivo and in vitro experiments. By binding to NLRP3, AS effectively inhibits the assembly and activation of the inflammasome. These findings suggest that AS is a promising inhibitor for PD driven by NLRP3 overactivation.

Ulcerative colitis (UC) is one of the most prevalent inflammatory bowel diseases and poses a serious threat to human health. Currently, safe and effective preventive measures are unavailable. In this study, the protective effects of asiaticoside (AS) on dextran sodium sulfate (DSS)-induced colitis in mice and the underlying molecular mechanism were investigated. In this experiment, colitis was induced in mice with DSS. Subsequently, the role of AS in colitis and its underlying mechanisms were examined using H&E staining, immunofluorescence staining, western blot, Elisa, FMT, and other assays. The results showed that AS significantly attenuated the related symptoms of DSS-induced colitis in mice. In addition, AS inhibited the activation of signaling pathways TLR4/NF-κB and MAPK reduced the release of inflammatory factors, thereby attenuating the inflammatory response in mice. AS administration also restored the permeability of the intestinal barrier by increasing the levels of tight junction-associated proteins (claudin-3, occludin, and ZO-1). In addition, AS rebalanced the intestinal flora of DSS-treated mice by increasing the diversity of the flora. AS can alleviate DSS-induced ulcerative colitis in mice by maintaining the intestinal barrier, thus inhibiting the signaling pathways TLR4/NF-κB and MAPK activation, reducing the release of inflammatory factors, and regulating intestinal microecology.