麻花秦艽-红景天药对不同比例配伍抗缺氧作用及机制研究

Study on Anti-hypoxia Effect and Mechanism of Gentiana Straminea Maxim.-Rhodiola Crenulata(Hook.f.et Thoms.)H.Ohba Combination of Different Proportions

目的筛选麻花秦艽-红景天药对抗缺氧的最佳配伍比例,联合体内、外实验及网络药理学探讨其抗缺氧作用及可能机制。方法将PC12细胞进行分组,通过Na2S2O4法、H2O2法及物理缺氧法建立细胞缺氧模型,对麻花秦艽-红景天药对抗缺氧配伍比例进行初步筛选;将80只SPF级雄性昆明小鼠随机分为空白组、模型组、阳性组、麻花秦艽-红景天不同配伍比例组(2∶8,3∶7,4∶6,5∶5,6∶4,7∶3,8∶2),每组8只。预先灌胃给药15 d后,除空白组外,其余各组均进行常压缺氧试验,记录各组小鼠生存时间并检测空白组、模型组、阳性组、麻花秦艽-红景天4∶6组及麻花秦艽-红景天3∶7组小鼠肺组织中的炎性细胞因子、MDA含量及SOD活力;通过网络药理学、分子对接技术对麻花秦艽-红景天药对抗缺氧机制进行初步探讨,并通过qPCR进行实验验证。结果体外实验研究发现,麻花秦艽-红景天药对具有较好的抗缺氧活性;体内实验结果显示,4∶6及3∶7配伍比例麻花秦艽-红景天药对可明显提高小鼠的存活时间,降低肺组织中NF-κB、IL-6及IL-1β及MDA含量,升高IL-10含量及SOD活力,且以麻花秦艽-红景天3∶7组疗效最佳;网络药理学研究发现,麻花秦艽-红景天药对抗缺氧的潜在活性成分可能为熊果酸、齐墩果酸、没食子乙酸乙酯等,核心靶点有SRC、PIK3CA、MAPK3等,其抗缺氧作用信号通路主要为PI3K-Akt、HIF-1等。qPCR结果显示,麻花秦艽-红景天可升高缺氧小鼠肺组织中的PI3K、Akt、mTOR及p62表达。分子对接验证显示核心靶点SRC、PIK3CA及p62与潜在活性成分齐墩果酸、山奈酚、没食子乙酸乙酯、槲皮素的结合活性较好。结论麻花秦艽-红景天药对具有抗缺氧活性,其通过抗炎、抗氧化应激及调控自噬发挥抗缺氧作用,可能与PI3K/Akt信号通路有关。

OBJECTIVE To screen the optimal compatibility ratio of Gentiana straminea Maxim.(G.S Maxim)and Rhodiola crenulata(Hook.f.et Thoms.)H.Ohba(R.crenulata),and explore its anti-hypoxia effect and possible mechanism through in vivo and in vitro experiments and network pharmacology.METHODS PC12 cells were divided into groups,and the cell hypoxia model was established by Na2S2O4,H2O2 and physical hypoxia methods,and the compatibility ratio of G.S Maxim-R.crenulata was initially screened.Eighty SPF male Kunming mice were randomly divided into blank group,model group,positive group,and different compatibility ratio groups of G.S Maxim-R.crenulata(2∶8,3∶7,4∶6,5∶5,6∶4,7∶3,8∶2),with 8 mice in each group.Gastric drug delivery in 15 d in advance,in addition to the blank group,the rest of the groups were normal pressure hypoxia experiments.Groups of mice survival time was recorded.Detected the levels of inflammatory cytokines,MDA content,and SOD activity in the lung tissues of mice in the blank group,model group,positive group,G.S Maxim-R.crenulata 4∶6 group and 3∶7 group.The anti-hypoxia mechanism of G.S Maxim-R.crenulata was investigated by network pharmacology and molecular docking,and verified by qPCR.RESULTS In vitro experiments showed that G.S Maxim-R.crenulata had better anti-hypoxia activity.In vivo experimental results showed that the combination ratio of G.S Maxim-R.crenulata with 4∶6 and 3∶7 could significantly improve the survival time of mice,reduce the contents of NF-κB,IL-6,IL-1βand MDA in lung tissue,and increase the content of IL-10 and SOD activity,and the effect of G.S Maxim-R.crenulata 3∶7 group was the best.Network pharmacological studies showed that the potential active components of G.S Maxim-R.crenulata in anti-hypoxia might be ursolic acid,oleanolic acid,and ethyl gallate,etc.The core targets included SRC,PIK3CA,MAPK3,etc.,and its anti-hypoxia signaling pathways mainly included PI3K-Akt,HIF-1,etc.The results of qPCR showed that G.S Maxim-R.crenulata could increase the expression of PI3K,Akt,mTOR and p62 in the lung tissue of hypoxic mice.Molecular docking verification showed that the core targets SRC,PIK3CA,and p62 had good binding activity with potential active components such as oleanolic acid,kaempferol,ethyl gallate and quercetin.CONCLUSION G.S Maxim-R.crenulata has anti-hypoxia activity,which may be related to PI3K/Akt signaling pathway through anti-inflammatory,anti-oxidative stress and regulation of autophagy.