目的 通過全細(xì)胞膜片鉗實驗,從心肌電生理層面探索丹紅注射液(DHI)治療烏頭堿誘導(dǎo)心律失常的可能機制。方法 基于人誘導(dǎo)多能干細(xì)胞衍生心肌細(xì)胞(hiPSC-CMs)膜片鉗技術(shù),即時給藥并觀察烏頭堿不同濃度(3、9、27 μmol·L^-1)對鈉通道、hERG鉀通道的抑制效果,進行造模條件的篩選;設(shè)置對照組、模型組(烏頭堿1 μmol·L^-1長時間造模)、DHI (3、9、27 μL·mL^-1)組,烏頭堿與DHI同時加藥,記錄動作電位幅度(APA)、放電頻率、動作電位幅度下降50 %時的時程(APD₅₀)和動作電位幅度下降90 %時的時程(APD₉₀),并以對照組為標(biāo)準(zhǔn),計算相對值;設(shè)置對照組、模型組、DHI (3 μL·mL^-1)組,烏頭堿與DHI同時加藥,觀察3~5 min,記錄鈉離子、hERG鉀離子、鈣離子電流密度。結(jié)果 給予即時藥物處理,3、9、27 μmol·L^-1的烏頭堿對鈉電流的抑制率分別為17.94 %、31.07 %、60.67 %,對hERG通道的抑制率分別為5.02 %、10.59 %、28.59 %;在短期內(nèi)給烏頭堿對hERG通道的抑制效果較為有限,而實際實驗中,長時間的高濃度烏頭堿孵育使鈉電流被完全抑制,導(dǎo)致離子通道功能受損,因此選擇烏頭堿1 μmol·L^-1長時間給藥制備模型。與對照組比較,模型組hiPSC-CMs相對APA、相對APD₅₀和相對APD₉₀顯著下降(P<0.01、0.001),相對放電頻率顯著升高(P<0.001);鈉電流在一定時間內(nèi)減小,抑制率為30.18 %;hERG鉀電流明顯減小,抑制率為72.33 %;鈣電流明顯增大,電流增大191.35 %。與模型組比較,DHI 3、9、27 μL·mL^-1組相對放電頻率顯著降低(P<0.05、0.001)、相對APD₉₀顯著升高(P<0.05、0.01),3 μL·mL^-1組相對APD₅₀顯著升高(P<0.001);DHI組鈉電流、hERG鉀電流的減小及鈣電流的增大均有一定緩解。結(jié)論 DHI可能可以通過影響鈉、鉀、鈣通道來緩解烏頭堿導(dǎo)致的心律失?，F(xiàn)象。

Objective To explore the possible mechanism of Danhong Injection (DHI) in the treatment of aconitine-induced arrhythmia from the perspective of cellular electrophysiology through whole-cell patch-clamp experiments. Methods Based on the patch-clamp technique of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), the inhibition effects of aconitine at different concentrations (3, 9, 27 μmol·L^-1) on sodium channel and hERG potassium channel were observed, and the modeling conditions were screened. The control group, model group (1 μmol·L^-1 aconitine for a long time) and DHI (3, 9, 27 μL·mL^-1) groups were set up, and aconitine and DHI were added at the same time. The action potential amplitude (APA), discharge frequency, time-domain of 50 % action potential decrease (APD₅₀) and time-domain of 90 % action potential decrease (APD₉₀) were recorded, and the relative values were calculated using the control group as the standard. The control group, model group and DHI (3 μL·mL^-1) group were set up, and aconitine and DHI were added at the same time, observed for 3-5 min, and the current densities of sodium ion, hERG potassium ion, and calcium ion were recorded. Results The inhibition rates of aconitine at 3, 9, and 27 μmol·L^-1 on sodium current were 17.94 %, 31.07 %, and 60.67 %, respectively, and the inhibition rates of hERG channel were 5.02 %, 10.59 %, and 28.59 %, respectively. The inhibition effect of aconitine on hERG channel in a short period of time was limited, while in the actual experiment, the long-term incubation of high concentration of aconitine completely inhibited the sodium current, resulting in the damage of ion channel function, so the model was prepared by 1 μmol·L^-1 aconitine for a long time. Compared with the control group, the relative APA, relative APD₅₀, and relative APD₉₀ of hiPSC-CMs in the model group were significantly decreased (P < 0.01, 0.001), and the relative discharge frequency was significantly increased (P < 0.001). The sodium current decreased within a certain time, with an inhibition rate of about 30.18 %. The hERG potassium current decreased significantly, with an inhibition rate of about 72.33 %. The calcium current increased significantly, with an increase of about 191.35 %. Compared with model group, the relative discharge frequency of DHI 3, 9, and 27 μL·mL^-1 groups was significantly decreased (P < 0.05, 0.001), and the relative APD 90 was significantly increased (P < 0.05, 0.01), and the relative APD₅₀ of 3 μL·mL^-1 group was significantly increased (P < 0.001). The decrease of sodium current, the decrease of hERG potassium current, and the increase of calcium current in the DHI group were alleviated to some extent. Conclusion DHI may alleviate aconitine-induced arrhythmias by influencing sodium, potassium, and calcium channels. potassium, and calcium channels.

R285.5

國家自然科學(xué)基金面上項目(81973698)