目的 探究理氣活血滴丸對(duì)大鼠心肌細(xì)胞H9C2缺氧/復(fù)氧（ H/R）損傷的保護(hù)作用及相關(guān)信號(hào)通路機(jī)制。方法 理氣活血滴丸175 mg·kg^-1 ig給予大鼠制備含藥血清，ig給予大鼠去離子水制備空白血清。將生長(zhǎng)狀態(tài)良好的H9C2細(xì)胞分為9組：對(duì)照[10%胎牛血清（ FBS）]組、單給空白血清（ 10%空白血清）組、模型（ 10% FBS＋H/R模型）組、空白血清（ 10%空白血清預(yù)處理12 h＋H/R模型）組、含藥血清（ 10%含藥血清預(yù)處理12 h＋H/R模型）組、shNC＋空白血清（ NC-shRNA感染＋10%空白血清預(yù)處理12 h＋H/R模型）組、shHIF-1α＋空白血清（ HIF-1α-shRNA感染＋10%空白血清預(yù)處理12 h＋H/R模型）組、shNC＋含藥血清（ NC-shRNA感染＋10%含藥血清預(yù)處理12 h＋H/R模型）組、shHIF-1α＋含藥血清（ HIF-1α-shRNA感染＋10%含藥血清預(yù)處理12 h＋H/R模型）組。重組shRNA腺病毒感染H9C2細(xì)胞24 h后，更換相應(yīng)培養(yǎng)基預(yù)處理12 h，隨后進(jìn)行H/R處理。酶聯(lián)免疫吸附測(cè)定法（ ELISA）檢測(cè)心肌酶[乳酸脫氫酶（ LDH）、肌酸激酶同工酶MB（ CKMB）、心肌肌鈣蛋白I（ cTnI）]漏出情況，CCK-8法檢測(cè)細(xì)胞活性，Annexin V-APC/7-AAD染色流式細(xì)胞術(shù)分析細(xì)胞凋亡情況，Western blotting檢測(cè)B細(xì)胞淋巴瘤/白血病-2（ Bcl-2）、Bcl-2相關(guān)X蛋白（ Bax）、裂解型半胱氨酸天冬氨酸蛋白酶3（ cleaved Casepase-3）、缺氧誘導(dǎo)因子-1α（ HIF-1α）、血紅素加氧酶1（ HO-1）蛋白表達(dá)情況，免疫熒光觀察HIF-1α亞細(xì)胞定位并行平均光密度（ AOD）分析。結(jié)果 與單給空白血清組比較，H/R后空白血清組H9C2心肌細(xì)胞的培養(yǎng)液中LDH、CKMB和cTnI含量顯著升高（ P＜0.01），細(xì)胞活力顯著下降（ P＜0.05）、凋亡率顯著增加（ P＜0.05），Bcl-2蛋白表達(dá)顯著降低（ P＜0.05），Bax、cleaved Casepase-3、HIF-1α、HO-1蛋白表達(dá)顯著升高（ P＜0.05），HIF-1α陽(yáng)性表達(dá)定位于細(xì)胞質(zhì)和細(xì)胞核，HIF-1α AOD顯著升高（ P＜0.05）。與空白血清組比較，理氣活血滴丸含藥血清預(yù)處理可使H/R后心肌細(xì)胞的心肌酶LDH、CK-MB、cTnI漏出顯著減少（ P＜0.05），細(xì)胞活力顯著升高（ P＜0.05）、凋亡率顯著降低（ P＜0.05），Bcl-2蛋白表達(dá)顯著升高（ P＜0.05），Bax、cleaved Casepase-3、HIF-1α、HO-1蛋白表達(dá)顯著降低（ P＜0.05），HIF-1α在細(xì)胞質(zhì)和細(xì)胞核都有表達(dá)，但主要定位于細(xì)胞核，HIF-1α AOD顯著降低（ P＜0.05）。重組腺病毒介導(dǎo)的HIF-1α-shRNA可削弱理氣活血滴丸含藥血清上述作用（ P＜0.05）。結(jié)論 理氣活血滴丸通過(guò)調(diào)節(jié)HIF-1α信號(hào)通路減輕H/R誘導(dǎo)的心肌細(xì)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

國(guó)家自然科學(xué)基金資助項(xiàng)目（82360965；82060770）