目的 基于鈣信號轉(zhuǎn)導(dǎo)探討穩(wěn)心顆?？剐穆墒С５目赡茏饔脵C(jī)制。方法 60只雄性SD大鼠隨機(jī)分為對照組、模型組及穩(wěn)心顆粒低、中、高劑量（1.34、2.68、5.36 g/kg）組和胺碘酮（35.7 mg/kg）組。各組大鼠連續(xù)預(yù)防性ig給藥21 d，對照組和模型組ig純水，末次給藥1 h后麻醉大鼠，尾iv烏頭堿以誘導(dǎo)心律失常。采用RM6240多道生理信號采集處理系統(tǒng)記錄大鼠心電表現(xiàn)，并進(jìn)行心電參數(shù)分析。采用蛋白質(zhì)免疫印跡法（Western blotting，WB）檢測心肌組織鈣/鈣調(diào)蛋白依賴性蛋白激酶II（calcium/calmodulin-dependent protein kinase II，CaMKII）、蘭尼堿受體2（ryanodine receptor 2，RyR2）和L-型鈣通道（L-type calcium channel，LTCC）的蛋白表達(dá)。采用瞬時轉(zhuǎn)染方法將編碼LTCC（α1dδ、α2δ、β2α）和綠色熒光蛋白（green fluorescent protein，GFP）的質(zhì)粒共轉(zhuǎn)染至人胚胎腎細(xì)胞HEK293上，予以穩(wěn)心顆粒含藥培養(yǎng)液干預(yù)，用鈣離子熒光探針（Fluo-4 AM）檢測細(xì)胞內(nèi)鈣離子濃度的變化、膜片鉗技術(shù)檢測細(xì)胞膜上LTCC電流變化。結(jié)果 尾iv烏頭堿后大鼠出現(xiàn)心律失常，與對照組比較，模型組大鼠心率顯著加快（P＜0.05），心律失常評分顯著升高（P＜0.01），CaMKⅡ和RYR2蛋白表達(dá)增加（P＜0.01），LTCC蛋白表達(dá)減少（P＜0.01）；與模型組比較，穩(wěn)心顆粒高劑量組和胺碘酮組心率顯著減慢（P＜0.05），心律失常評分降低（P＜0.01），CaMKⅡ和RYR2蛋白表達(dá)減少（P＜0.05、0.01），LTCC蛋白表達(dá)增加（P＜0.05、0.01）。穩(wěn)心顆粒在0.78～12.50 g/L對細(xì)胞的促增殖作用最強(qiáng)（P＜0.05、0.01）；穩(wěn)心顆粒能增加LTCC電流峰值和電流密度，導(dǎo)致電壓依賴性通道激活曲線發(fā)生負(fù)向偏移、電壓依賴性通道失活曲線發(fā)生正向偏移。結(jié)論 穩(wěn)心顆?？赡芡ㄟ^影響鈣信號轉(zhuǎn)導(dǎo)相關(guān)蛋白表達(dá)并改變L-型鈣通道的門控動力學(xué)發(fā)揮抗心律失常效應(yīng)。

Objective To investigate the possible mechanism of anti-arrhythmic action of Wenxin Granules (穩(wěn)心顆粒) based on calcium signal transduction. Methods In animal experiment, 60 male SD rats were randomly divided into control group, model group, Wenxin Granules low, medium and high dose (1.34, 2.68, 5.36 g/kg) groups and amiodarone (35.7 mg/kg) group. The rats in each group were given continuous prophylactic administration for 21 d, and the control group and model group were given pure water. The rats were anesthetized 1 h after the last administration and injected with aconitine through the tail vein to induce arrhythmia. The electrocardiograph (ECG) performance of rats was recorded by RM6240 physiological signal acquisition and processing system, and the ECG parameters were analyzed. Western blotting (WB) was used to detect calcium/calmodulin-dependent protein kinase II (CaMKII), ryanodine receptor 2 (RyR2) and L-type calcium channel (LTCC) protein expression in myocardial tissue. In the cell experiment, the plasmid encoding LTCC (α1δ, α2δ, β2α) and green fluorescent protein (GFP) was transfected into HEK293 cells by transient transfection method and interfered with Wenxin Granules, the change of intracellular calcium ion concentration was detected by calcium ion fluorescence probe (Fluo-4 AM) and the change of LTCC current on cell membrane was detected by patch clamp technique. Results: Arrhythmia occurred in rats after being injected with aconitine through the tail vein. Compared with the control group, the heart rate of the model group was faster (P < 0.05), the arrhythmia score was higher (P < 0.01), the expression of CaMKⅡ and RYR2 protein was increased (P < 0.01), and the expression of LTCC protein was decreased (P < 0.01). Compared with model group, the heart rate of Wenxin Granules high-dose group and amiodarone group significantly slowed down (P < 0.05), arrhythmia score was decreased (P < 0.01), protein expressions of CaMKⅡand RYR2 decreased (P < 0.05, 0.01), and protein expression of LTCC increased (P < 0.05, 0.01). At the concentration of 0.78—12.50 g/L, Wenxin Granules had the strongest effect on cell proliferation (P < 0.05, 0.01).Wenxin Granules could increase the current peak value and current density of LTCC, resulting in negative deviation of voltage-dependent channel activation curve and positive deviation of voltage-dependent channel inactivation curve.Conclusion Wenxin Granules may exert antiarrhythmic effects by influencing the expression of calcium signal transduction related proteins and changing the gating dynamics of LTCC.

R285.5

國家自然科學(xué)基金項(xiàng)目（82004329）;天津市教委科研計劃項(xiàng)目（2024KJ020）;天津中醫(yī)藥大學(xué)新教師科研啟動項(xiàng)目“雛鷹計劃”項(xiàng)目（KG240215）;國家中醫(yī)藥管理局全國名老中醫(yī)藥專家傳承工作室建設(shè)項(xiàng)目“杜武勛全國名老中醫(yī)藥專家傳承工作室”（國中醫(yī)藥人教函 [2022] 75號）;天津市中醫(yī)藥管理局天津市名中醫(yī)傳承工作建設(shè)項(xiàng)目“杜武勛天津市名中醫(yī)傳承工作室”