目的 探究理气活血滴丸对大鼠心肌细胞H9C2缺氧/复氧（ H/R）损伤的保护作用及相关信号通路机制。方法 理气活血滴丸175 mg·kg^-1 ig给予大鼠制备含药血清，ig给予大鼠去离子水制备空白血清。将生长状态良好的H9C2细胞分为9组：对照[10%胎牛血清（ FBS）]组、单给空白血清（ 10%空白血清）组、模型（ 10% FBS＋H/R模型）组、空白血清（ 10%空白血清预处理12 h＋H/R模型）组、含药血清（ 10%含药血清预处理12 h＋H/R模型）组、shNC＋空白血清（ NC-shRNA感染＋10%空白血清预处理12 h＋H/R模型）组、shHIF-1α＋空白血清（ HIF-1α-shRNA感染＋10%空白血清预处理12 h＋H/R模型）组、shNC＋含药血清（ NC-shRNA感染＋10%含药血清预处理12 h＋H/R模型）组、shHIF-1α＋含药血清（ HIF-1α-shRNA感染＋10%含药血清预处理12 h＋H/R模型）组。重组shRNA腺病毒感染H9C2细胞24 h后，更换相应培养基预处理12 h，随后进行H/R处理。酶联免疫吸附测定法（ ELISA）检测心肌酶[乳酸脱氢酶（ LDH）、肌酸激酶同工酶MB（ CKMB）、心肌肌钙蛋白I（ cTnI）]漏出情况，CCK-8法检测细胞活性，Annexin V-APC/7-AAD染色流式细胞术分析细胞凋亡情况，Western blotting检测B细胞淋巴瘤/白血病-2（ Bcl-2）、Bcl-2相关X蛋白（ Bax）、裂解型半胱氨酸天冬氨酸蛋白酶3（ cleaved Casepase-3）、缺氧诱导因子-1α（ HIF-1α）、血红素加氧酶1（ HO-1）蛋白表达情况，免疫荧光观察HIF-1α亚细胞定位并行平均光密度（ AOD）分析。结果 与单给空白血清组比较，H/R后空白血清组H9C2心肌细胞的培养液中LDH、CKMB和cTnI含量显著升高（ P＜0.01），细胞活力显著下降（ P＜0.05）、凋亡率显著增加（ P＜0.05），Bcl-2蛋白表达显著降低（ P＜0.05），Bax、cleaved Casepase-3、HIF-1α、HO-1蛋白表达显著升高（ P＜0.05），HIF-1α阳性表达定位于细胞质和细胞核，HIF-1α AOD显著升高（ P＜0.05）。与空白血清组比较，理气活血滴丸含药血清预处理可使H/R后心肌细胞的心肌酶LDH、CK-MB、cTnI漏出显著减少（ P＜0.05），细胞活力显著升高（ P＜0.05）、凋亡率显著降低（ P＜0.05），Bcl-2蛋白表达显著升高（ P＜0.05），Bax、cleaved Casepase-3、HIF-1α、HO-1蛋白表达显著降低（ P＜0.05），HIF-1α在细胞质和细胞核都有表达，但主要定位于细胞核，HIF-1α AOD显著降低（ P＜0.05）。重组腺病毒介导的HIF-1α-shRNA可削弱理气活血滴丸含药血清上述作用（ P＜0.05）。结论 理气活血滴丸通过调节HIF-1α信号通路减轻H/R诱导的心肌细胞凋亡。

Objective To investigate the protective effect of Liqi Huoxue Dripping Pill on hypoxia/reoxygenation (H/R) injury in rat H9C2 myocardial cells and the related signaling pathway mechanisms. Methods The rats were given 175 mg·kg^-1 of Liqi Huoxue Dropping Pills by intragastric administration to prepare the drug-containing serum, and deionized water was given by intragastric administration to prepare the blank serum. H9C2 cells in good growth condition were divided into nine groups: the control group [10% fetal bovine serum (FBS)], the single administration blank serum group (10% blank serum), the model group (10% FBS + H/R model), the blank serum pretreatment group (10% blank serum pretreatment for 12 h + H/R model), the drug-containing serum pretreatment group (10% drug-containing serum pretreatment for 12 h + H/R model), the shNC + blank serum group (NC-shRNA infection + 10% blank serum pretreatment for 12 h + H/R model), the shHIF-1α + blank serum group (HIF-1α-shRNA infection + 10% blank serum pretreatment for 12 h + H/R model), the shNC + drug-containing serum group (NC-shRNA infection + 10% drug-containing serum pretreatment for 12 h + H/R model), and the shHIF-1α + drug-containing serum group (HIF-1α-shRNA infection + 10% drugcontaining serum pretreatment for 12 h + H/R model). After 24 h of infection with recombinant shRNA adenovirus, the H9C2 cells were replaced with the corresponding culture medium for 12 h of pretreatment, followed by H/R treatment. The leakage of myocardial enzymes [lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), and cardiac troponin I (cTnI)] was detected by enzyme-linked immunosorbent assay (ELISA), cell viability was detected by CCK-8 method, apoptosis was analyzed by Annexin VAPC/7-AAD staining flow cytometry, and the protein expression of B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3, HIF-1α, and heme oxygenase 1 (HO-1) was detected by Western blotting. Immunofluorescence was used to observe the subcellular localization of HIF-1α and the average optical density (AOD) was analyzed. Results Compared with the single administration blank serum group, the content of LDH, CK-MB, and cTnI in the culture medium of H9C2 myocardial cells in the blank serum group after H/R was significantly increased (P < 0.01), cell viability was significantly decreased (P < 0.05), the apoptosis rate was significantly increased (P < 0.05), Bcl-2 protein expression was significantly decreased (P < 0.05), and the expression of Bax, cleaved caspase-3, HIF-1α, and HO-1 proteins was significantly increased (P < 0.05). HIF-1α positive expression was located in the cytoplasm and nucleus, and the AOD of HIF-1α was significantly increased (P < 0.05). Compared with the blank serum group, pretreatment with Liqi Huoxue Dropping Pills drug-containing serum could significantly reduce the leakage of myocardial enzymes LDH, CK-MB, and cTnI in H9C2 myocardial cells after H/R (P < 0.05), significantly increase cell viability (P < 0.05), significantly reduce the apoptosis rate (P < 0.05), significantly increase Bcl-2 protein expression (P < 0.05), and significantly decrease the expression of Bax, cleaved caspase-3, HIF-1α, and HO-1 proteins (P < 0.05). HIF-1α was expressed in both the cytoplasm and nucleus, but mainly located in the nucleus, and the AOD of HIF-1α was significantly decreased (P < 0.05). Recombinant-adenovirus-mediated HIF-1α- shRNA could weaken the above effects of Liqi Huoxue Dropping Pills drug-containing serum (P < 0.05). Conclusion Liqi Huoxue Dripping Pills alleviating H/R-induced cardiocyte apoptosis by regulating the HIF-1α signaling pathway.

R285.5

国家自然科学基金资助项目（82360965；82060770）