目的 對(duì)比熟地黃水提取物（RRPAE）與生地黃水提取物（DRRAE）治療肝缺血再灌注損傷的藥效和機(jī)制，以期為臨床患者更優(yōu)的治療選擇提供科學(xué)依據(jù)。方法 將48只C57雄性小鼠隨機(jī)分為8組（每組6只）：假手術(shù)組，模型組，RRPAE低、中、高劑量（2.5、5.0、10.0 g·kg^-1）組，DRRAE低、中、高劑量（2.5、5.0、10.0 g·kg^-1）組。ig給藥，假手術(shù)組和模型組ig 0.9%氯化鈉溶液，每天1次，連續(xù)1周；各組小鼠于末次給藥2 h后行手術(shù)建立小鼠肝缺血再灌注損傷模型，假手術(shù)組僅開(kāi)腹不缺血，再灌注后6 h取材。檢測(cè)各組小鼠肝臟、脾臟系數(shù)；蘇木素-伊紅染色觀察各組小鼠肝臟組織病理學(xué)改變；試劑盒法檢測(cè)小鼠血清丙氨酸轉(zhuǎn)氨酶（ALT）、天冬氨酸轉(zhuǎn)氨酶（AST）、乳酸脫氫酶（LDH）、一氧化氮（NO）水平；試劑盒法檢測(cè)小鼠肝臟超氧化物歧化酶（SOD）、NO、丙二醛（MDA）水平；Western blotting法檢測(cè)各組小鼠肝臟組織中鐵死亡相關(guān)蛋白轉(zhuǎn)鐵蛋白（Transferrin）、溶質(zhì)載體家族7成員11 （SLC7A11）、溶質(zhì)載體家族39成員14 （SLC39A14）、poly結(jié)合蛋白2（PCBP2）的表達(dá)水平。培養(yǎng)BRL肝細(xì)胞，用50、100、150 mg·mL^-1的RRPAE處理1 h （對(duì)照組不加藥）后，加野百合堿（500 μmol·L^-1）或埃拉斯?。?0 μmol·L^-1）處理24 h，試劑盒法分別檢測(cè)細(xì)胞亞鐵/總鐵離子以及谷胱甘肽水平。結(jié)果 與模型組比較，經(jīng)過(guò)RRPAE或DRRAE預(yù)給藥后，給藥組小鼠的肝臟、脾臟系數(shù)出現(xiàn)下降趨勢(shì)，其中RRPAE低、高劑量組肝臟系數(shù)差異顯著（P＜0.05、0.001）；病理染色結(jié)果顯示模型組肝臟出現(xiàn)明顯的缺血-充血區(qū)和肝細(xì)胞壞死，經(jīng)過(guò)RRPAE與DRRAE給藥后肝細(xì)胞狀態(tài)均有所恢復(fù)，且前者的效果明顯優(yōu)于后者。生化結(jié)果顯示，與模型組相比，RRPAE可顯著降低血清中肝損傷指標(biāo)AST、ALT、LDH水平，升高NO含量；顯著降低肝臟MDA水平，升高SOD和NO水平（P＜0.05、0.01） ；而DRRAE給藥后對(duì)上述肝損傷及氧化應(yīng)激相關(guān)指標(biāo)的恢復(fù)作用整體并不顯著。Western blotting實(shí)驗(yàn)結(jié)果顯示，與模型組比較，RRPAE能夠顯著抑制促進(jìn)鐵死亡相關(guān)蛋白Transferrin、SLC39A14的表達(dá)，上調(diào)抑制鐵死亡相關(guān)蛋白SLC7A11、PCBP2的表達(dá)（P＜0.05、0.001） ，而DRRAE對(duì)鐵死亡相關(guān)蛋白的調(diào)控并不明顯。與模型組比較，RRPAE可上調(diào)野百合堿或鐵死亡激動(dòng)劑埃拉斯汀誘導(dǎo)的肝細(xì)胞培養(yǎng)基中亞鐵/總鐵離子以及谷胱甘肽水平（P＜0.05、0.01、0.001）。結(jié)論 熟地黃緩解肝缺血再灌注損傷效果更好，其作用機(jī)制可能與調(diào)控Transferrin、SLC7A11、SLC39A14、PCBP2蛋白的表達(dá)，抑制小鼠鐵死亡進(jìn)程有關(guān)。

Objective Based on comparing the efficacy and mechanism of aqueous extracts of Rehmanniae Radix Praeparata (RRPAE) and drying Rehmannia root (DRR) for the treatment of hepatic ischemia-reperfusion injury (HIRI), to provide a better therapeutic option for clinical patients. Method Totally 48 mice were randomly divided into eight groups: the sham group, the model group, the low, medium, and high dose groups of RRPAE (2.5, 5.0 and 10.0 g·kg^-1), and the low, medium, and high dose groups of DRRAE (2.5, 5.0 and 10.0 g·kg^-1), with six mice in each group. After one week of pre-administration of different doses of RRPAE or DRRAE by ig, the mouse HIRI model was established, and the HE staining was used to observe the histopathological changes in the livers of the mice in each group. The kits were used to detect the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), nitric oxide (NO) levels. The kits were used to detect mouse liver superoxide dismutase (SOD), nitric oxide (NO), and malondialdehyde (MDA) levels. Western blotting was used to detect ferroptosis-associated proteins in mouse liver tissues of each group: Transferrin, solute carrier family 7 member 11 (SLC7A11), solute carrier family 39 member 14 (SLC39A14), and poly rC binding protein 2 (PCBP2). After culturing and treating BRL hepatocytes with monocrotaline (MCT, 500 μmol·L^-1) or ferroptosis agonist erastin (10 μmol·L^-1), different doses of RRPAE (50, 100, and 150 μg·mL^-1) were administered for 24 h, and then the cellular ferrous iron/ total iron ions and glutathione content were detected. Results Compared with the model group, after pre-administration of RRPAE or DRRAE, the liver and spleen coefficients of mice in the treatment group showed a downward trend, with significant differences in liver coefficients in the low and high dose RRPAE groups (P＜0.05, 0.001). HE staining results showed significant ischemia-reperfusion region and hepatocellular necrosis in the HIRI group, and the hepatocellular status was recovered after both RRPAE and DRRAE administration, and the former was significantly better than the latter. The biochemical results showed that compared with the model group, the serum levels of AST, ALT and LDH, which were indicators of liver injury, decreased significantly, while the level of NO increased significantly; the level of hepatic MDA decreased significantly, while the levels of SOD and NO increased significantly (P＜0.05, 0.01), and the modulation effect of DRR on the above indicators of liver injury and oxidative stress was not significant. The results of Western blotting experiments showed that the RRPAE was able to significantly inhibit the expression of Transferrin and SLC39A14-related ferroptosis-promoting proteins, and down-regulate the expression of SLC7A11 and PCBP2-related ferroptosis-suppressing proteins (P＜0.05, 0.001), whereas the DRRAE did not significantly regulate the above iron-death-related proteins. At the same time, RRPAE directly inhibited the ferroptosis process of hepatocytes induced by monocrotaline or ferroptosis agonist erastin, which was accompanied by an increase in ferrous iron/total iron ions and glutathione content in the cell culture medium (P＜0.05, 0.01, and 0.001). Conclusion The medicinal type of DRR with better effect in alleviating hepatic ischemia-reperfusion injury is RRPAE, and its mechanism of action may be related to the regulation of Transferrin, SLC7A11, SLC39A14, and PCBP2 protein expression and inhibition of ferroptosis process in mice.

R285.5

國(guó)家自然科學(xué)基金面上項(xiàng)目（82274186）