갯질경이 용매분획물의 3T3-L1전지방세포에서의 지방생성억제 효과

Antiadipogenic Activity of Solvent-partitioned Fractions from Limonium tetragonum in 3T3-L1 Preadipocytes

Abstract

갯질경이(Limonium tetragonum)는 질경이과에 속하는 여러해살이 풀로 습지에 자생하는 염생식물의 일종이며, 항산화, 항종양 및 간보호 효능이 있는 것으로 알려져 있다. 본 연구에서는 갯질경이 추출물로부터 용매 극성에 따라 분획한 분획물($H_2O$, n-BuOH, 85% aq. MeOH 및 n-Hexane)을 이용하여 지방세포내 중성지방 생성 및 지방세포 분화조절 인자 발현에 미치는 영향을 검토하였다. 마우스 유래 지방전구세포3T3-L1을 지방세포로 분화하여 Oil Red O염색법으로 지방 세포 분화정도를 확인한 결과, 갯질경이 분획물에 의해 지방세포의 형성이 농도의존적으로 억제되었다. 또한 지방생성조절에 관여하는 전사인자 $PPAR{\gamma}$, $C/EBP{\alpha}$ 및 SREBP-1c의 발현을 mRNA와 단백질 수준에서 확인한 결과 갯질경이 분획물 처리시 지방세포 분화 인자의 발현이 유의적으로 감소하였다. 지방세포 분화에 관여하는 것으로 알려진 MAPK 신호 전달 경로를 확인한 결과 갯질경이 분획물 처리군에서 p38, ERK 및 JNK의 인산화가 억제되었다. 용매 분획물중에서 $H_2O$ 및 n-Hexane 분획물이 가장 우수한 지방생성 억제활성을 나타내었는데 이는, 분획물 중 페놀 또는 지방 유도체에 의한 것으로 사료된다. 본 연구 결과로부터 갯질경이 분획물의 MAPK 신호전달 경로 억제를 통한 항비만 효과를 확인하였으며, 나아가 건강기능성 식품 소재로서의 개발 가능성이 기대된다.

Limonium tetragonum, an edible halophyte that grows on salt marshes in Korea, is thought to possess various health benefits (e.g., antioxidant, antitumor, and hepatoprotective). In the present study, different solvent partitioned subfractions, water ($H_2O$), buthanol (n-BuOH), 85% aqueous methanol (85% aq. MeOH), and hexane (n-hexane), from crude extract of L. tetragonum were tested for their ability to prevent adipogenesis in differentiating 3T3-L1 preadipocytes. The treatment of differentiating 3T3-L1 preadipocytes with L. tetragonum subfractions (LTFs) resulted in suppressed adipogenesis and reduced expression of adipogenesis-related transcription factors such as peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$), CCAATT/enhancer-binding protein alpha ($C/EBP{\alpha}$), and sterol regulatory element-binding protein 1c (SREBP-1c) at both mRNA and protein levels. In addition, the LTF treatment notably decreased the levels of phosphorylated p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK) pathway in association with $PPAR{\gamma}$-linked adipogenesis. Among all the tested LTFs, $H_2O$ and n-hexane were the most effective in lowering lipid accumulation and regulating the adipocyte differentiation via $PPAR{\gamma}$ pathway. Taken together, the results indicated that the $H_2O$ and n-hexane LTFs contain bioactive compounds that may exhibit significant antiadipogenesis activity by downregulation of the $PPAR{\gamma}$ pathway and inactivation of the MAPK signal pathway in 3T3-L1 preadipocytes.

Fig. 1. Cytotoxic effect of LTFs on 3T3-L1 cells. The data represent the mean ± SD of three separate experiments.

Fig. 2. Effect of LTFs on the lipid accumulation of differentiated 3T3-L1 adipocytes depicted by Oil red O staining and the quantification of the stain bound to lipid droplets. The data represent the mean ± SD of three separate experiments.

Fig. 3. mRNA expression levels of key adipogenic factors from PPARγ in differentiated mature 3T3-L1 adipocytes following treatment of LTFs (H₂O; n-BuOH; 85% aq. MeOH; n-Hexane) assayed by RT-PCR. β-actin was used as a housekeeping protein control. The data represent the mean ± SD of three separate experiments. ^a-eMeans with the different letters are significantly different by Duncan’s multiple range test. (significant as compared to control. *p<0.05).

Fig. 4. Effect of LTFs (H₂O; n-BuOH; 85% aq. MeOH; n-Hexane) on the protein levels of the PPARγ pathway transcription actors analyzed by Western blotting. The protein levels were showed as protein bands and effects were observed as the band density. β-actin was used as a housekeeping protein control. Data presented as percentage of untreated differentiated control cell group normalized against β-actin.

Fig. 5. Effect of LTFs (H₂O; n-BuOH; 85% aq. MeOH; n-Hexane) on the protein levels of the phosphorylated (p-) MAPK pathway proteins p38, ERK and JNK analyzed by Western blotting. The protein levels were showed as protein bands and effects were observed as the band density. β-actin was used as a housekeeping protein control. Data presented as percentage of untreated differentiated control cell group normalized against β-actin.