Investigation of the effect of B. spergulifolius extracts on insulin-resistant 3T3-L1 adipocytes

Diabetes mellitus (DM) is a metabolic disease with chronic hyperglycemia characterized by metabolic outcomes due to insufficient insulin secretion and/or insulin effect defect. DM often causes cardiovascular diseases and various complications on the eye, kidney and nervous system. Type 2 DM (T2DM) accounts for approximately 90% of the total DM. Therefore, it is very important to investigate new therapeutic approaches for T2DM and alternative, natural agents that target molecules in potential signal pathways. Medicinal plants are very significant resources in the research of alternative new drug active ingredients. Bolanthus spergulifolius (B. spergulifolius) is one of the genera of the family Caryophyllaceae. In the present study, it was aimed to investigate the potential anti-diabetic effects in-vitro of B. spergulifolius extracts on 3T3-L1 adipocytes. B. spergulifolius were extracted with methanol (MeOH), ethylacetate (EA) and Aqueous and evaluated for their total phenolic content (TPC) by Folin-Ciocalteu method. B. spergulifolius extracts showing highly TPC (Aqueous< MeOH< EA) and their different concentrations were caried out on preadipocytes differentiated in to mature 3T3-L1 adipocytes to investigate their half-maximal (50%) inhibitory concentration (IC50) by using MTT assay. The IC50 concentrations of MeOH, EA and Aqueous extracts were observed as 305.7 ± 5.583 µg/mL, 567.4 ± 3.008 µg/mL and 418.3 ± 4.390 µg/mL and were used for further experiments. A live/dead assay further confirmed the cytotoxic effect of MeOH, EA and Aqueous extracts (respectively, 69.75 ± 1.70%, 61.75 ± 1.70%, 70 ± 4.24% and for all P< 0.05). Also, effects of extracts on lipid accumulation in mature 3T3-L1 adipocytes were evaluated by Oil-Red O staining assay. The extracts effectively decreased lipid-accumulation compared to untreated adipocytes (for all P< 0.05). Morever, effect of extracts on apoptosis regulated by the Bax and Bcl-2 was investegated by qRT-PCR. The extracts significantly induced apoptosis by up-regulating pro-apoptotic Bax expression but down-regulating anti-apoptotic Bcl-2 gene expression compared to untreated adipocytes (for all P< 0.05). The Glut-4 expression linked with insulin resistance was determined by qRT-PCR, Western-blot and Immunofluorescence staining. In parallel, expression of Glut-4 in adipocytes treated with extracts was significantly lower compared to untreated adipocytes (for all P< 0.05). Extracts significantly supressed cell migration after 30 h of wounding in a scratch-assay (for all P< 0.05). Cell morphology and diameter were further evaluated by phase-contrast microscopy, Scanning electron microscope (SEM), Immunofluorescence (with F-Actin) and Giemsa staining. The adipocytes treated with extracts partially lost spherical morphology and showed smaller cell-diameter compared to untreated adipocytes (for all P< 0.05). In conclusion, these results suggest that extracts of B. spergulifolius causes to an induce apoptosis, a decrease lipid-accumulation, wound healing, Glut-4 level and migth contribute to the reducing of insulin-resistance in DM. Key Words: Bolanthus spergulifolius, diabetes mellitus, insulin-resistant, 3T3-L1 adipocytes, Glucose transporter-4, anti-diabetic