DSGOST powder was prepared as previously described (24). In brief, herbal components of DSGOST were mixed and subsequently extracted by hot water (10-fold volume), comprising 1 g Angelica radix, 1 g Cinnamomi cortex, 1 g Paeoniae root, 1 g Akebia root, 0.67 g Asarum, 0.67 g Glycyrrhiza, 1.67 g Zizyphus jujuba, 0.67 g Evodia fruit and 1.33 g Ginger root. The dried mixture was stored at −80°C until use. The composition of the DSGOST herbal prescription is shown in Table I.

Human umbilical vein endothelial cells (HUVECs), human dermal microvascular endothelial cells (HDMECs) and pericytes were purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA). HUVECs and HDMECs were cultured in endothelial medium supplemented with 5% fetal bovine serum and 1% endothelial cell growth supplement (ScienCell Research Laboratories, Inc.). Pericytes from human brain microvessels were cultured in pericyte medium (ScienCell Research Laboratories, Inc.) supplemented with 5% fetal bovine serum (ScienCell Research Laboratories, Inc.), 1% pericyte growth supplement and 1% penicillin/streptomycin solution.

For cold-mediated contraction, endothelial cells and pericytes were pretreated with DSGOST (100 µg/ml) for 30 min, and subsequently cultured at physiological temperature (37°C) or a cooler temperature (25°C) for 30 min. In case of ET-1-mediated pericyte contraction, instead of the cooler condition (i.e. at 25°C), pericytes were incubated with ET-1 (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). Following the incubation at the cooler temperature with ET-1, cells were harvested for protein extraction using iced radioimmunoprecipitation assay (RIPA) buffer. The extracted proteins were mixed with sample buffer and boiled at 100°C for 10 min. The boiled samples were separated using 8–12% SDS-PAGE according to their molecular weights, and then transferred to a nitrocellulose membrane for western blotting. The proteins were blotted using the following antibodies: Rabbit anti-phospho-cofilin (Ser3) monoclonal (cat. no. 3313; 1:1,000), rabbit anti-phospho-LIM domain kinase (LIMK)1 (Thr508)/LIMK2 (Thr505) polyclonal (cat. no. 3841; 1:1,000), rabbit anti-testis-specific kinase 1 (TESK1) monoclonal (cat. no. 4655; 1:1,000), rabbit anti-PDXP monoclonal (cat. no. 4686; 1:1,000) and rabbit anti-phospho-myosin light chain 2 (Ser19; cat. no. 3671; 1:1,000) antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse-anti-RhoA-GTP monoclonal antibody (cat. no. 26904; 1:500; NewEast Biosciences, Malvern, PA, USA).

Six-week-old BALB/c mice were purchased from Jungang Lab Animal, Inc. (Seoul, Korea). Mice were housed under conditions of a constant temperature (24±2°C) with 12 h-light/12 h-dark cycle. All experimental procedures were approved by the Kyung Hee University Institutional Animal Care and Use Committee (KHU-IACUC). Mice were randomly grouped and administered orally with drinking water or DSGOST (10 mg/kg). After 30 min, mice were anesthetized using Avertin solution which was prepared by mixing 2,2,2-tribromoethanol (Sigma-Aldrich; Merck Millipore) and 2-methyl-2-butanol, tertiary amyl alcohol 99% (Sigma-Aldrich; Merck Millipore), according to the manufacturer's protocol. Subsequently, their tails were soaked under cool (25±2°C) or warm (37±2°C) water for 30 min, using a heat block to maintain constant temperature. The 10 mm length of the segment from extremities of the tail were fixed in formaldehyde and embedded in paraffin. The paraffin blocks were sectioned at 15 µm and mounted on slide glasses. For hematoxylin and eosin (H&E) staining, the prepared slides were immersed in H&E solution for staining the nuclei and the cytoplasm. The stained sections were photographed at a magnification of 40x, and subsequently the area of the vessels from each group was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Immunohistochemistry was performed using a Vextastain^® ABC-AP kit, according to the manufacturer's protocol (Vector Laboratories, Burlingame, CA, USA). Rabbit anti-α2c-AR polyclonal antibody (1:100) and rabbit anti-CD31 polyclonal antibody (1:100; Abcam, Cambridge, UK) were used, and the histological expression patterns of α2c-AR and CD31 were analyzed. Following staining with each of the antibodies, the sections were photographed at a magnification of 40x.

Statistical analysis was performed using SPSS V22.0 (IBM SPSS, Armonk, NY, USA). The differences of the means between the groups were analyzed using one-way analysis of variance and a Student's t-test. P<0.05 was used to indicate a statistically significant difference.

Since pericytes cause vessel contraction that is important for the processes of RP (1,25), whether treatment with DSGOST inhibited contraction of either cold (25°C)- or ET-1-exposed pericytes was first examined. DSGOST enhanced RhoA activation and increased the levels of ROCK1, TESK1 and PDXP in pericytes exposed to the cold, but not to ET-1, while slightly reducing the levels of LIMK1/2, cofilin and MLC for the two different conditions (Fig. 1). Therefore, DSGOST activation of the RhoA/ROCK/TESK1/PDXP pathway appeared to be unique to the cold-exposed pericytes.

Based on our previous work on the mitigative effects of DSGOST on the cold response in ECs (24), whether DSGOST affects the cold-induced RhoA pathway in ECs was subsequently investigated. DSGOST inhibited RhoA activation, the phosphorylation of LIMK1/2 and MLC, and reduced the level of ROCK1, with no effect on TESK1 and PDXP in cold-exposed ECs (Fig. 2).

To investigate DSGOST efficacy on cold-mediated vessel contraction, the vessel area was measured following incubation under each condition. DSGOST treatment rescued cold-induced vessel contraction and appeared to markedly promote vasodilation (Fig. 3). Thus, our data suggested that DSGOST may be useful for relieving cold-induced vessel contraction.

As increases in α2c-AR protein expression and the anterograde translocation of this protein to the plasma membrane occurs when cells are exposed to cooler temperatures (12–14), this protein was selected for the current investigation. In the immunohistochemical analysis, although α2c-AR was evidently detected in mouse tail vessels at 25°C, DSGOST treatment reduced its induction in the vessel (Fig. 4). Therefore, the oral administration of DSGOST was demonstrated to be effective at reducing cold-induced vessel contraction.