Hyoscyamus albus nortropane alkaloids reduce hyperglycemia and hyperinsulinemia induced in HepG2 cells through the regulation of SIRT1/NF-kB/JNK pathway

Background Chronic superphysiological glucose and insulin concentrations are known to trigger several tissue and organ failures, including insulin resistance, oxidative stress and chronic low-grade inflammation. Hence, the screening for molecules that may counteract such conditions is essential in current existing therapeutic strategies, thereby the use of medicinal plant derivatives represents a promising axis in this regard. Methods In this study, the effect of a selected traditional medicinal plant, Hyoscyamus albus from which, calystegines have been isolated, was investigated in an experimental model of hyperinsulinemia and hyperglycemia induced on HepG2 cells. The mRNA and protein expression levels of different insulin signaling, gluconeogenic and inflammatory pathway- related molecules were examined. Additionally, cell viability and apoptosis, oxidative stress extent and mitochondrial dysfunctions were assayed using flow cytometric and qRT-PCR techniques. Results Treatment of IR HepG2 cells with calystegines strongly protected the injured cells from apoptosis, oxidative stress and mitochondrial integrity loss. Interestingly, nortropane alkaloids efficiently regulated the impaired glucose metabolism in IR HepG2 cells, through the stimulation of glucose uptake and the modulation of SIRT1/Foxo1/G6PC/mTOR pathway, which is governing the hepatic gluconeogenesis. Furthermore, the alkaloidal extract restored the defective insulin signaling pathway, mainly by promoting the expression of Insr at the mRNA and protein levels. What is more, treated cells exhibited significant mitigated inflammatory response, as evidenced by the modulation and the regulation of the NF- κB/JNK/TLR4 axis and the downstream proinflammatory cytokines recruitment. Conclusion Overall, the present investigation demonstrates that calystegines from Hyoscyamus albus provide cytoprotection to the HepG2 cells against insulin/glucose induced insulin resistance and apoptosis due to the regulation of SIRT1/Foxo1/G6PC/mTOR and NF-κB/JNK/TLR4 signaling pathways.

Extraction and fractionation of subproteome from root tips of Hyoscyamus albus

Identification and quantification of different metabolites under stress, especially protein, is a vital way to understand plant adaptation mechanism. We established an efficient protein extraction method from the tiny amount (100 mg) of root tips of non-model medicinal plant Hyoscyamus albus, using bead-beating cell disruption, TRIzol extraction, and sequential chemical protein solubilization. H. albus is very well known for biosynthesized of different secondary metabolites like hyoscyamine, tropane alkaloids and scopolamine. Our method is rational for sample preparation even in small-scale proteomics of recalcitrant tissue and allows proficient, reproducible and impurity-free protein extraction. This method allows high-quality 2DE in mini-gel format (25 µg of protein/gel) for hydrophilic and hydrophobic sub-proteomes and is compatible to high-sensitive matrix-assisted laser/desorption ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF) mass spectrometry (MS). A protocol using TRIzol is more effective and reproducible to sequential chemical extraction of both hydrophilic and hydrophobic membrane proteins. We also demonstrated cell disrupted together with dithiothreitol (DTT) and polyvinylpolypyrrolidone (PVPP) is more useful to prevent polymerization of the phenolic compound than commonly used added DTT and PVPP with TRIzol reagent. Despite the unavailability of genomic sequence database, the efficacy of the protocol was also confirmed by MS/MS ion searches. J Bangladesh Agril Univ 17(4): 430–436, 2019