目的 探討小檗堿對(duì)心肌缺血再灌注損傷大鼠線粒體自噬及PTEN誘導(dǎo)激酶1（PTEN induced putative kinase 1，PINK1）/帕金森病蛋白（Parkin）通路的影響。方法 建立心肌缺血再灌注損傷大鼠模型，隨機(jī)分組為模型組、小檗堿低、高劑量（75、150 mg/kg）組，自噬抑制劑三甲基腺嘌呤（3-MA，100 mmol/L）組、小檗堿+3-MA（150 mg/kg+100 mmol/L）組，每組12只，另取12只正常大鼠設(shè)為假手術(shù)組。分組處理后，超聲檢測(cè)大鼠左室功能，記錄左室收縮末期內(nèi)徑（LVESD）、左室舒張末期內(nèi)徑（LVEDD）、左心射血分?jǐn)?shù)（LVEF）、左室短軸縮短率（FS）；三苯基氯化四氮唑（TTC）染色檢測(cè)各組大鼠心肌梗死面積，酶聯(lián)免疫吸附實(shí)驗(yàn)（ELISA）檢測(cè)各組大鼠血清中磷酸肌酸激酶同工酶（CK-MB）、肌鈣蛋白I（cTnI）水平；HE染色觀察大鼠心肌組織病理變化；透射電鏡觀察心肌細(xì)胞超微結(jié)構(gòu)及線粒體自噬并分析線粒體損傷評(píng)分；蛋白免疫印跡法檢測(cè)各組大鼠心肌組織PINK1、Parkin蛋白及微管輕鏈蛋白3B（LC3B）、線粒體自噬受體p62（p62）、泛素特異性蛋白酶30（USP30）蛋白表達(dá)。結(jié)果 與假手術(shù)組比較，模型組大鼠心肌組織病理?yè)p傷嚴(yán)重，線粒體腫脹及空泡化損傷較多，線粒體損傷評(píng)分、心肌梗死面積、LVEDD、LVESD、CK-MB、cTnI水平及PINK1、Parkin、LC3B、p62蛋白表達(dá)升高（P<0.05），LVEF及FS、USP30蛋白表達(dá)降低（P<0.05）。與模型組比較，3-MA組大鼠心肌組織及線粒體病理?yè)p傷加重，LVEF、FS、PINK1、Parkin、LC3B、p62蛋白表達(dá)降低（P<0.05），線粒體損傷評(píng)分、心肌梗死面積、LVEDD、LVESD、CK-MB、cTnI水平、USP30蛋白表達(dá)升高（P<0.05）；小檗堿低、高劑量大鼠心肌組織及線粒體病理?yè)p傷減輕，LVEF、FS、PINK1、Parkin、LC3B、p62、USP30蛋白表達(dá)升高（P<0.05），線粒體損傷評(píng)分、心肌梗死面積、LVEDD、LVESD、CK-MB、cTnI水平降低（P<0.05）。小檗堿+3-MA組大鼠上述各項(xiàng)指標(biāo)均與小檗堿高劑量組變化趨勢(shì)相反，且有統(tǒng)計(jì)學(xué)差異（P<0.05）。結(jié)論 小檗堿可能通過(guò)激活PINK1/Parkin/P62/LC3B通路促進(jìn)線粒體自噬，升高USP30表達(dá)，減少異常自噬，緩解心肌缺血再灌注損傷。

Objective To investigate the effects of berberine on mitochondrial autophagy and phosphatase and tensin homology deleted on chromosome ten (PTEN)-induced putative kinase 1 (PINK1)/Parkin pathway in rats with myocardial ischemia-reperfusion (MIR). Methods MIR rat model was established and randomly divided into model group, low-dose berberine group (75 mg/kg), high-dose berberine group (150 mg/kg), autophagy inhibitor trimethyladenine (3-MA, 100 mmol/L), berberine + autophagy inhibitor group (150 mg/kg + 100 mmol/L), with 12 rats in each group, and another 12 rats were set as Sham operation group. After grouping and treatment, left ventricular function was detected by echocardiography, left ventricular end systolic diameter (LVESD), left ventricular end diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF) and left ventricular short axis shortening (FS) were recorded. The infarct size was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and the levels of creatine kinase isoenzyme (CK-MB) and troponin I (cTnI) in serum were detected by enzyme-linked immunosorbent assay (ELISA). HE staining was used to observe the pathological changes of myocardium. The ultrastructure and mitochondrial autophagy of cardiomyocytes were observed by transmission electron microscopy (TEM), and mitochondrial damage score was analyzed. The protein expressions of PINK1, Parkin, microtubule associated protein 1 light chain 3B (LC3B), mitochondrial autophagy receptor p62 and ubiquitin specific protease 30 (USP30) were detected by Western blotting. Results Compared with those in the Sham operation group, the pathological damage of myocardial tissue in the model group was serious, and the swelling and vacuolation of mitochondria were more serious, the mitochondrial injury score, myocardial infarction area, LVEDD, LVESD, levels of CK-MB and cTnI, the protein expression levels of PINK1, Parkin, LC3B and p62 were higher (P<0.05), but LVEF, the protein expression levels of FS and USP30 were lower (P<0.05). Compared with those in the model group, the pathological damage of myocardial tissue and mitochondria in 3-MA group was aggravated, LVEF, FS, the protein expression levels of PINK1, Parkin, LC3B and p62 were lower (P<0.05), the mitochondrial injury score, myocardial infarction area, LVEDD, LVESD, levels of CK-MB and cTnI and the protein expression level of USP30 were higher (P<0.05). The pathological damage of myocardial tissue and mitochondria in rats in low and high dose berberine groups was alleviated, LVEF, FS, the protein expression levels of PINK1, Parkin, LC3B, p62 and USP30 were higher (P<0.05), the mitochondrial damage score, myocardial infarction area, LVEDD, LVESD, levels of CK-MB and cTnI were lower (P < 0.05). The changes of the above indexes in berberine + 3-MA group were contrary to those in high-dose berberine group, and the difference was statistically significant (P<0.05). Conclusion Berberine may promote mitochondrial autophagy by activating PINK1/Parkin/p62/LC3B pathway, increase USP30 expression, reduce abnormal autophagy and alleviate MIR injury.

R285

國(guó)家自然科學(xué)基金資助項(xiàng)目（81603114）