key: cord-0927392-mxhhos0l
authors: Xia, Yi-Teng; Hu, Wei-Hui; Wu, Qi-Yun; Ting-Xia Dong, Tina; Duan, Ran; Xiao, Jian; Li, Shao-ping; Qin, Qi-Wei; Wang, Wen-Xiong; Wah-Keung Tsim, Karl
title: The herbal extract deriving from aerial parts of Scutellaria baicalensis shows anti-inflammation and anti-hypoxia responses in cultured fin cells from rabbit fish
date: 2020-07-29
journal: Fish Shellfish Immunol
DOI: 10.1016/j.fsi.2020.07.050
sha: 68562448e47707c7bd1ba53bf0de325692a0d8dd
doc_id: 927392
cord_uid: mxhhos0l

A new cell line derived from dorsal fin of rabbit fish Siganus fuscescens was developed and characterized. The cell line was isolated from the dorsal fin, named as rabbit fish fin (RFF) cell line, and which was sub-cultured for 50 cycles since the development. This cell line was tested for growth in different temperatures and serum concentrations, and the best growing condition was at 20% serum at 28 °C. In cultured RFF cells, amplification of 18S rRNA from genomic DNA and immunostaining of cellular cytokeratin confirmed the proper identity of S. fuscescens fish. After 30th passage of cultures, the cells were exposed to challenge of inflammation, triggered by LPS, and hypoxia, mimicked by CoCl(2). Cultured RFF cells showed robust sensitive responses to inflammation and hypoxia in directing the expressions of cytokines and hypoxia inducible factor-1α (HIF-1α). The water extract of aerial part of Scutellaria baicalensis (SBA) has been shown in rabbit fish to prevent inflammation. Here, we extended this notion of testing the efficacy of SBA extract in the developed cultured RFF cells. Application of SBA extract inhibited the expression of LPS-induced inflammatory cytokines, i.e. IL-1β, IL-6, as well as the signaling of NF-κB. The application of CoCl(2) in cultured RFF cells triggered the hypoxia-induced cell death and up regulation of HIF-1α. As expected, applied SBA extract in the cultures prevented the hypoxia-induced signaling. Our results show the established RFF cell line may be served as an ideal in vitro model in drug screening relating to inflammation and hypoxia. Additionally, we are supporting the usage of SBA herbal extract in fish aquaculture, which possesses efficacy against inflammation and hypoxia.

fuscescens using microElute Genomic DNA kit (Omega Bio-tek, Norcross, GA). The 20% FBS (Fig. 2A) . The growth was at peak after 6 days of culture. Serum at 10% or 30% in 244 the medium could not support the proper growth of RFF cells. Thus, 20% FBS in medium 245 was employed for culture at 28°C, and which should be used with 6 days of culture.

The effect of SBA extract to relieve inflammation 248 In order to test inflammatory response of cultured RFF cells, LPS was applied here to induce To further demonstrate potential NF-κB and IL-6 suppression mediating by SBA extract, the 274 transfected RFF cells with pNF-κB-Luc or pIL-6-Luc were used here. In transfected RFF cells, 275 the activities of pNF-κB-Luc and pIL-6-Luc were robustly induced by LPS, and which were 276 suppressed markedly by pre-treatment of SBA extract (Fig. 4 B&C) . A maximal suppression 277 was revealed at 0.1 to 0.2 mg/mL of SBA extract. Again, dexamethasone suppressed the 278 LPS induction, robustly. The translocation of p65 subunit of NF-κB in LPS-induced RFF cells 279 was probed here. As expected, application of LPS in cultures induced the nuclear 280 translocation of NF-kB p65 subunit (Fig. 5A) . Around 45% of NF-κB p65 subunit was 281 translocated into nuclear (Fig. 5B) . The translocation of NF-κB p65 subunit was inhibited by (Fig. 6C) . Having 30% of the temperature-induced cell death, application of SBA 297 extract protected the cell in dose-dependent manner (Fig. 6D) . At low dose of SBA (0.1 298 mg/mL), the cell survival could be restored to over 80%.

To cope with hypoxia, HIF is the critical one, and which is requiring a binding between an 

Transcriptional regulation of the gilthead 1. A new cell line divided from Siganus fuscescens was established and named as RFF cell

The RFF cell exhibited sensitive responses to LPS-induced inflammation and hypoxia environment

The aerial part of S. baicalensis extract showed anti-inflammatory and anti-hypoxia effects on RFF cell, indicating the potential application as an aquaculture feeding additive

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.