L-肉碱对庆大霉素致子代豚鼠毛细胞损伤干预作用

目的观察围产期L-肉碱对庆大霉素暴露致子代豚鼠听力损伤及耳蜗毛细胞凋亡的影响。方法将18只孕中期豚鼠按体重随机分为3组,从孕51～57 d,对照组腹膜内注射生理盐水100 mg/kg、庆大霉素组腹膜内注射庆大霉素100 mg/kg,L-肉碱组腹膜内注射庆大霉素100 mg/kg同时以饮水方式给予L-肉碱100 mg/mL;出生后14 d,检测子代豚鼠畸变产物耳声发射(distortion product otoacoustic emission,DPOAE)改变,应用原位末端转移酶标记技术(TUNEL)染色和免疫组化检测毛细胞凋亡情况。结果围产期庆大霉素暴露致子代豚鼠DPOAE幅值在频率1、2、4、6、8 kHz下分别为(8.38±2.28)、(12.56±3.22)、(10.46±1.98)、(12.22±1.72)、(17.73±2.16)dB,L-肉碱组在相同频率下DPOAE幅值分别为(14.26±1.47)、(20.98±2.39)、(19.23±2.15)、(22.56±1.41)、(29.13±3.14)dB;庆大霉素暴露致子代豚鼠耳蜗毛细胞凋亡率为34.6%,L-肉碱可降低毛细胞凋亡率至15.7%(P<0.05)。结论 L-肉碱对庆大霉素致子代豚鼠耳毒性具有干预作用。     

Role of L-carnitine in the prevention of acute liver damage induced by carbon tetrachloride in rats

BACKGROUND AND AIM: Lipid peroxidation is the most important mechanism in the pathogenesis of acute liver damage with carbon tetrachloride (CCl4). L-carnitine may prevent lipid peroxidation and thus may protect against liver damage. In the present study we investigated the protective effect of L-carnitine in experimental acute liver damage induced by CCl4. METHODS: Fifty rats were allocated to five equal groups. The first group was the control (group 1), the second group received an intraperitoneal CCl4 injection for 3 days (group 2), and the third group received a 50 mg/kg subcutaneous L-carnitine injection for 4 days, beginning a day before CCl4 injection. The CCl4 injection was continued for 3 days in the concerned group (group 3). Group 4 was given a CCl4 injection for 7 days and group 5 received a 50 mg/kg subcutaneous L-carnitine injection for 8 days, beginning a day before CCl4 injection. This group continued to receive a CCl4 injection for 7 days. Rats in groups 2 and 3 were killed on the fifth day. Rats in groups 1, 4 and 5 were killed on the ninth day. Plasma and liver tissue malondialdehyde (MDA) levels, glutathione peroxidase (GSH-Px) activity and liver enzyme levels were studied. Histopathological investigations were conducted. RESULTS: Liver tissue MDA levels decreased significantly in group 3 compared to group 2 (P<0.001). Liver tissue MDA levels in group 5 decreased significantly in comparison to those of group 4 (P<0.001). Liver tissue GSH-Px activity in group 5 was significantly lower than that in group 4 (P<0.05). There were no significant differences between groups 3 and 4 regarding GSH-Px activity (P>0.05). Steatosis, inflammation and necrosis in group 3 were significantly reduced when compared to group 2 (P<0.01). Fibrosis development was not identified in groups 2 and 3. Steatosis in group 5 was significantly lower than that in group 4 (P<0.05) and there were no significant differences between groups 4 and 5 with regards to inflammation and necrosis (P>0.05). Mild fibrosis development was identified in groups 4 and 5 but the difference between the groups was not significant. CONCLUSION: It appears that L-carnitine has a protective effect in the early stage of experimental acute liver damage induced by CCl4. As the toxic effect or damage continues, its effect lessens.     

当归补血汤联合左旋卡尼汀治疗肾性贫血的临床观察

目的:观察当归补血汤联合左旋卡尼汀治疗肾性贫血的临床疗效。方法:选取2017年1月至2018年12月安徽医科大学附属宿州医院就诊的肾性贫血患者110例作为研究对象,按照随机数字表法随机分为对照组和观察组,每组55例。2组均给予常规治疗,在此基础上,对照组给予左旋卡尼汀治疗,观察组给予当归补血汤联合左旋卡尼汀治疗,均连续治疗3个月,比较2组患者的贫血相关指标、肾功能、中医证候积分及临床疗效。结果:观察组患者总有效率为94.55%,对照组为78.18%,差异有统计学意义(χ²=6.25,P<0.05);2组患者红细胞(RBC)、血红蛋白(Hb)和红细胞比容(HCT)水平均高于治疗前(均P<0.05),且治疗后观察组RBC、Hb和HCT水平显著高于对照组(均P<0.05);2组治疗后肾功能指标及中医证候积分均较治疗前下降(均P<0.05),且治疗后观察组明显低于对照组(均P<0.05)。结果:当归补血汤联合左旋卡尼汀能显著提高肾性贫血患者RBC和Hb水平,改善贫血相关症状体征及肾功能,临床效果较好。     

L-carnitine对缺氧/复氧新生大鼠心肌细胞能量生成及CPT-Ⅱ mRNA表达的影响

目的 探讨L-肉毒碱(L-carnitine,L-Car)对缺氧／复氧培养新生大鼠心肌细胞能量生成的影响及其机制。方法 以原代培养新生大鼠心肌细胞建立缺氧／复氧损伤模型,高效液相色谱检测单纯缺氧／复氧以及L-Car预处理缺氧／复氧心肌细胞ATP、ADP、AMP生成改变;RT-PCR方法检测心肌细胞肉毒碱脂酰基转移酶-Ⅱ(carnitine palmitoyltransferase-Ⅱ,OPT-Ⅱ)mRNA表达。结果 与正常对照组比较,培养心肌细胞缺氧24h复氧0、2、4、8h后,心肌细胞ATP、ADP均明显下降。与单纯缺氧／复氧组比较,L-Car预处理组心肌细胞缺氧／复氧后ATP含量明显增高;培养心肌细胞缺氧24h后,CPT-ⅡmRNA表达除复氧2h有所上升外,复氧4、8h表达渐次降低。L-Car预处理使缺氧24h复氧4h心肌细胞CPT-Ⅱ mRNA表达呈浓度依赖性增高,50nmol／L处理组CPT-Ⅱ mRNA表达显著高于0nmol／L处理组。结论 心肌细胞缺氧／复氧过程中能量生成明显降低,其损伤可能与三羧酸循环中呼吸酶OPT-Ⅱ表达降低有关;L-Car对培养仔鼠心肌细胞缺氧复氧损伤具有显著保护作用,其机制可能是与L-Car从转录调控水平改善CPT-Ⅱ合成有关。     

左卡尼汀对脓毒症心肌损伤的保护作用

目的：观察左卡尼汀在脓毒症合并心肌损伤患者中的保护作用。方法：将29例严重脓毒症患者分为治疗组（15例）和对照组（14例）。治疗组在综合治疗基础上加左卡尼汀治疗,对照组采用抗感染等综合治疗措施。在研究开始前及研究d3、d7抽血检测心肌肌钙蛋白I（CTnI）、N末端脑钠肽前体（NT-proBNP）,比较两组ICU住院死亡率。结果：①治疗前CTnI水平：治疗组（3.24±2.8）ng/ml,对照组（2.48±1.69）ng/ml;NT-proBNP水平：治疗组（13711.93±6260.07）pg/ml,对照组（11790.43±8879.37）pg/ml。两组间CTnI及NT-proBNP均较正常值升高,组间比较差异均无统计学意义（P＞0.05）。②治疗d3 CTnI水平：治疗组（1.47±1.14）ng/ml,对照组（1.4±1.01）ng/ml;NT-proBNP水平：治疗组（12194.73±7515.39）pg/ml,对照组（17202.29±7325.23）pg/ml。两组CTnI水平均有下降,对照NT-proBNP水平升高,但CTnI及NT-proBNP组间比较差异均无统计学意义（P＞0.05）。③治疗d7 CTnI水平：治疗组（0.61±0.78）ng/ml,对照组（1.90±1.90）ng/ml;NT-proBNP水平：治疗组（7413.47±6277.00）pg/ml,对照组（10958.57±6651.45）pg/ml。两组CTnI及NT-proBNP水平均有下降,治疗组的CTnI及NT-proBNP水平均较对照组下降明显,组间比较CTnI差异有统计学意义（P＜0.05）,NT-proBNP差异无统计学意义（P＞0.05）。④死亡率：两组均有3例死亡,ICU住院死亡率分别为20.00%VS 21.43%,死亡率差异无统计学意义（P＞0.05）。结论：左卡尼汀能够减轻脓毒症患者的心肌损伤,改善心功能,具有心肌保护作用。     

左卡尼汀对异丙肾上腺素诱导大鼠心肌肥厚的保护作用及机制研究

目的观察左卡尼汀(L-carnitine)对异丙肾上腺素(Iso)诱导的大鼠心肌肥厚的保护作用,并探讨其心肌保护作用的可能机制。方法应用异丙肾上腺素(Iso)制备SD大鼠心肌肥厚模型。40只健康大鼠,随机分成正常对照组(空白组)、异丙肾上腺素组(Iso组)、左卡尼汀组(L-carni-tine组)和普萘洛尔组(propranolol组)各10只。除空白组外各组大鼠均皮下注射(sc)Iso 10 d制成心肌肥厚模型。大鼠饲养到期后,检测心脏功能和心脏重量指数;制备心室肌石蜡切片,行Masson染色,光镜下观察心肌形态;测定大鼠心肌组织中的游离脂肪酸(FFA)、乳酸(LAC)和羟脯氨酸(Hpy)水平。分离心肌细胞进行线粒体膜电位(MMP)的测定。结果相比Iso组,L-carnitine组大鼠心脏功能改善,心脏重量指数降低;光镜下观察:心肌细胞肥大程度较Iso组减轻,心肌纤维排列整齐,胶原纤维增生较少;脂肪酸(FFA)、乳酸(LAC)和羟脯氨酸(Hpy)水平增高;心肌线粒体膜电位升高。结论应用L-carnitine在一定程度上可以抑制心肌肥厚的发展,其机制可能与改善能量代谢和细胞线粒体的功能有关。     

左卡尼汀对新兵越野跑后血清葡萄糖和游离脂肪酸的影响

目的观察左卡尼汀对新兵空腹5000 m越野跑前后血清葡萄糖(Glu)和游离脂肪酸(FFA)的影响,为探讨军训低血糖的预防方法提供依据。方法选入伍男性新兵54名,随机分为服药组(n=24)和对照组(n=30)。服药组于空腹越野跑5000 m前10 d开始口服左卡尼汀,1.0g,3次/d;对照组服安慰剂。检测跑前10 min与跑后即刻和4 h时Glu、糖原磷酸化酶同功酶BB(Glycogen phosphorylase isoenzyme BB,GPBB)、FFA和心型脂肪酸结合蛋白(heart-type fatty acid-bind-ing protein,H-FABP)的变化,并与对照组比较。结果服药组越野跑前10 min Glu为(4.65±0.32)mmol/L,越野跑后的即刻为(5.66±1.02)mmol/L,越野跑后4 h为(4.49±0.66)mmol/L,显著高于对照组〔(3.78±0.52)mmol/L(、4.36±0.48)mmol/L、(3.89±0.46)mmol/L〕(P<0.01)。服药组在越野跑后4 h,FFA为(126.27±56.77)μmol/L,显著高于对照组〔(99.78±18.76)μmol/L〕(P<0.01)。服药组越野跑后H-FABP无显著变化(P>0.05);2个组在越野跑后4 h,GPBB虽显著高于越野跑前10 min(P<0.01),但服药组在越野跑后即刻和4 h均显著低于对照组(P<0.01)。结论左卡尼汀可提高越野跑前后Glu水平,减少越野跑后FFA的下降,对预防新兵空腹越野跑所致的低血糖具有一定的保护作用。     

左旋卡尼汀对维持性血液透析患者微炎症、贫血和心功能的影响

目的：观察左旋卡尼汀对维持性血液透析（MHD）患者微炎症、贫血和心功能的影响,探讨左旋卡尼汀对MHD患者的治疗作用。方法：选择已排除急性感染及其他活动性疾病、透析龄超过6个月的MHD患者42例,随机分为治疗组（22例）和对照组（20例）。每次透析结束后,治疗组给予静脉注射左旋卡尼汀2g,对照纽注射等量的生理盐水。观察治疗前及治疗4个月后患者炎症介质、营养状况、贫血和心脏结构与心功能。结果：与治疗前相比,治疗组的超敏C反应蛋白（Hs．CRP）、白介素（IL-6）显著下降,血红蛋白（Hb）、红细胞压积（Hct）显著升高,左心室收缩末内径（LVDs）,左心室射血分数（LVEF）明显改善;与对照组比较,治疗组的Hs-CRP、IL-6、Hb、Hct、LVDs、左心室舒张末内径（LVDd）、LVEF差异显著。结论：左旋卡尼汀可改善MHD微炎症状态、贫血和心功能,可作为MHD患者的一种长期治疗药物。     

左旋卡尼汀联合CrkL降低缺氧复氧诱导的心肌细胞损伤

目的研究左旋卡尼汀联合CT10激酶调节子样蛋白(CrkL)降低缺氧复氧(H/R)诱导的心肌细胞损伤的作用。方法利用H/R损伤心肌细胞H9c2,使用左旋卡尼汀处理。细胞计数试剂盒8(CCK-8)、流式细胞术、Western blot分别检测细胞的增殖、凋亡和细胞核相关抗原Ki-67(Ki-67)、增殖细胞核抗原(PCNA)、B细胞淋巴瘤/白血病2(Bcl-2)、Bcl-2相关X蛋白(Bax)、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、核因子κB(NF-κB)、CrkL水平。在细胞H9c2中转染pcDNA-CrkL,使用H/R处理或H/R+左旋卡尼汀处理。采用上述方法检测细胞增殖、凋亡等。结果与对照组比较,H/R组H9c2细胞的活力、Ki-67、PCNA、Bcl-2、CrkL蛋白表达量明显降低,细胞凋亡率、Bax蛋白水平及炎症因子TNF-α、IL-1β、NF-κB的蛋白水平显著升高(P0.05)。与H/R组比较,左旋卡尼汀明显增加H/R诱导的H9c2细胞活力及Ki-67、PCNA、Bcl-2、CrkL蛋白表达量,显著降低细胞凋亡率、Bax蛋白水平及TNF-α、IL-1β、NF-κB蛋白水平(P0.05)。CrkL过表达明显提高H/R诱导的H9c2细胞活力和Ki-67、PCNA、Bcl-2蛋白表达量,显著降低细胞凋亡率、Bax蛋白水平及TNF-α、IL-1β、NF-κB蛋白水平(P0.05)。与单独使用左旋卡尼汀或CrkL过表达比较,左旋卡尼汀联合CrkL过表达明显提高H9c2细胞的活力和Ki-67、PCNA、Bcl-2蛋白表达量,显著降低细胞凋亡率、Bax蛋白水平及TNF-α、IL-1β、NF-κB蛋白水平(P0.05)。结论左旋卡尼汀联合CrkL可以促进缺氧复氧诱导的心肌细胞增殖,降低细胞凋亡和炎症反应,从而保护心肌细胞。     

L-carnitine alleviates cardiac microvascular dysfunction in diabetic cardiomyopathy by enhancing PINK1-Parkin-dependent mitophagy through the CPT1a-PHB2-PARL pathways

Aim

To explore the beneficial effects of L-carnitine on cardiac microvascular dysfunction in diabetic cardiomyopathy from the perspectives of mitophagy and mitochondrial integrity.

Methods

Male db/db and db/m mice were randomly assigned to groups and were treated with L-carnitine or a solvent for 24 weeks. Endothelium-specific PARL overexpression was attained via adeno-associated virus serotype 9 (AAV9) transfection. Adenovirus (ADV) vectors overexpressing wild-type CPT1a, mutant CPT1a, or PARL were transfected into endothelial cells exposed to high glucose and free fatty acid (HG/FFA) injury. Cardiac microvascular function, mitophagy, and mitochondrial function were analyzed by immunofluorescence and transmission electron microscopy. Protein expression and interactions were assessed by western blotting and immunoprecipitation.

Results

L-carnitine treatment enhanced microvascular perfusion, reinforced endothelial barrier function, repressed the endothelial inflammatory response, and maintained the microvascular structure in db/db mice. Further results demonstrated that PINK1-Parkin-dependent mitophagy was suppressed in endothelial cells suffering from diabetic injury, and these effects were largely alleviated by L-carnitine through the inhibition of PARL detachment from PHB2. Moreover, CPT1a modulated the PHB2-PARL interaction by directly binding to PHB2. The increase in CPT1a activity induced by L-carnitine or amino acid mutation (M593S) enhanced the PHB2-PARL interaction, thereby improving mitophagy and mitochondrial function. In contrast, PARL overexpression inhibited mitophagy and abolished all the beneficial effects of L-carnitine on mitochondrial integrity and cardiac microvascular function.

Conclusion

L-carnitine treatment enhanced PINK1-Parkin-dependent mitophagy by maintaining the PHB2-PARL interaction via CPT1a, thereby reversing mitochondrial dysfunction and cardiac microvascular injury in diabetic cardiomyopathy.