Department of Pharmacy, The Third Xiangya Hospital, Central South University, 138# Tong Zi Po Road, Changsha, 410013, Hunan, People's Republic of China.
Department of Pharmacy, Shaoxing Seventh People's Hospital, Shaoxing, Zhejiang, China.
BMC Complement Altern Med. 2019 Jun 20;19(1):138. doi: 10.1186/s12906-019-2560-2.
Oxymatrine (OM), a quinolizidine alkaloid extracted from a herb Sophorae Flavescentis Radix, has been used to treat liver fibrotic diseases. However, the mechanism of its anti-fibrosis effects is still unclear. TGF-β/Smad signaling and miR-195 have been proved to paly an important role in hepatic stellate cells (HSCs) activation and liver fibrosis. In this study, we investigated whether OM could inhibit HSCs activation through TGF-β1/miR-195/Smads signaling or not.
First, the effects of OM on HSC-T6 in different concentrations and time points were tested by MTT assay. We choose three appropriate concentrations of OM as treatment concentrations in following experiment. By Quantitative Real-time PCR and Western Blot, then we investigated the effect of OM on miR-195, Smad7 and α-SMA's expressions to prove the correlation between OM and the TGF-β1/miR-195/Smads signaling. Last, miR-195 mimic and INF-γ were used to investigate the relation between miR-195 and OM in HSC activation.
Our results showed that the proliferation of HSC was significantly inhibited when OM concentration was higher than 200 μg/mL after 24 h, 100 μg/mL after 48 h and 10 μg/mL after 72 h. The IC of OM after 24, 48 and 72 h were 539, 454, 387 μg/mL respectively. OM could down-regulate miR-195 and α-SMA (P < 0.01), while up-regulate Smad7 (P < 0.05). In HSC-T6 cells transfected with miR-195 mimic and pretreated with OM, miR-195 and α-SMA were up-regulated (P < 0.05), and Smad7 was down-regulated (P < 0.05) .
Given these results, OM could inhibit TGF-β1 induced activation of HSC-T6 proliferation in a dose-dependent and time-dependent manner to some extent. We proved that OM inhibited HSC activation through down-regulating the expression of miR-195 and up-regulating Smad7.
氧化苦参碱（OM）是从苦参中提取的一种喹诺里西啶生物碱，已被用于治疗肝纤维化疾病。然而，其抗纤维化作用的机制尚不清楚。TGF-β/Smad 信号和 miR-195 已被证明在肝星状细胞（HSCs）激活和肝纤维化中发挥重要作用。在这项研究中，我们研究了 OM 是否通过 TGF-β1/miR-195/Smads 信号通路抑制 HSCs 激活。
首先，通过 MTT 测定法检测 OM 在不同浓度和时间点对 HSC-T6 的影响。我们选择三个合适的 OM 浓度作为后续实验的处理浓度。通过定量实时 PCR 和 Western Blot，研究 OM 对 miR-195、Smad7 和 α-SMA 表达的影响，以证明 OM 与 TGF-β1/miR-195/Smads 信号之间的相关性。最后，使用 miR-195 模拟物和 INF-γ 研究 miR-195 在 HSC 激活中与 OM 的关系。
我们的结果表明，当 OM 浓度高于 24 h 时 200μg/mL、48 h 时 100μg/mL 和 72 h 时 10μg/mL 时，HSC 的增殖明显受到抑制。OM 在 24、48 和 72 h 时的 IC 分别为 539、454 和 387μg/mL。OM 可以下调 miR-195 和 α-SMA（P<0.01），同时上调 Smad7（P<0.05）。在转染 miR-195 模拟物并经 OM 预处理的 HSC-T6 细胞中，miR-195 和 α-SMA 上调（P<0.05），Smad7 下调（P<0.05）。
鉴于这些结果，OM 可以在一定程度上剂量依赖性和时间依赖性地抑制 TGF-β1 诱导的 HSC-T6 增殖的激活。我们证明 OM 通过下调 miR-195 的表达和上调 Smad7 抑制 HSC 激活。
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