腺苷酸激活蛋白酶介导糖尿病大鼠心肌线粒体自噬的机制与TSC2的关系研究

目的:探讨腺苷酸激活蛋白酶(AMP-activated protein kinase,AMPK)介导糖尿病大鼠心肌线粒体自噬的机制与结节性硬化复合物-2(tuberous sclerosis complex-2,TSC2)的关系。方法:成年雄性SD大鼠30只,随机分为对照组、模型组、模型+AMPK激动剂AICAR组(AICAR组)。采用腹腔注射链脲佐菌素建立大鼠糖尿病大鼠模型。AICAR组大鼠连续皮下注射AICAR 12周。采用超声诊断仪检测所有大鼠的心功能:左室舒张末期容积(left ventricular end-diastolic volume,LVEDV)、左室收缩末期容积(left ventricular end-systolic volume,LVESV)、左室射血分数(left ventricular ejection fraction,LVEF)、心率(heart rate,HR)、舒张早期血流峰值速度(peak velocity of early diastolic flow,E)、舒张晚期血流峰值速度(peak velocity of late diastolic flow,A)、E/A。取心肌组织,采用免疫共沉淀法检测AMPK和TSC2的结合水平;Western blot检测AMPK、TSC2、p-TSC2、LC3-Ⅱ、LC3-Ⅰ、Beclin-1和P62蛋白的表达水平。结果:AICAR组LVEDV、LVEF、E和E/A均高于模型组,而A低于模型组(P=0.004,P=0.001,P=0.000,P=0.011,P=0.027);AICAR组AMPK和TSC2蛋白表达水平高于模型组(0.71±0.06 vs.0.36±0.09,P=0.003;0.80±0.02 vs.0.40±0.05,P=0.017);AICAR组结合水平高于模型组(0.49±0.09 vs.0.23±0.03,P=0.002);AICAR组p-TSC2蛋白水平高于模型组(1.48±0.07 vs.0.92±0.07,P=0.029);AICAR组LC3-Ⅱ/LC3-Ⅰ、Beclin-1和P62均高于模型组(P<0.05)。结论:AMPK介导糖尿病大鼠心肌线粒体自噬的机制可能与其对TSC2的磷酸化作用有关。

自噬在非酒精性脂肪性肝病发生发展中的作用

非酒精性脂肪性肝病(NAFLD)是以弥漫性肝细胞脂肪变性为主要特征的临床病理综合征,包括单纯性脂肪性肝病以及由其演变的脂肪性肝炎、脂肪性肝纤维化和肝硬化。原有的“两次打击”学说不能完全解释其发病机制,涉及多种因素的多重打击学说提供了更全面的描述,包括脂质毒性、炎症反应、纤维化和基因表达改变。自噬是溶酶体对胞浆成分降解的一种代谢途径。自噬参与该病多种发病机制,导致细胞脂质堆积、炎症反应和纤维化。本篇综述总结了Unc-51样自噬激活激酶1、腺苷酸激活蛋白酶、哺乳动物雷帕霉素复合物1与自噬的关系,并详细描述了自噬如何参与非酒精性脂肪性肝病的发生发展。

Effects of 95%ethanol extract of Aquilaria sinensis leaves on hyperglycemia in diabetic db/db mice

The leaves of Aquilaria sinensis（Lour.） Gilg have been used in folk medicine for the treatment of diabetes in Guangdong Province,China.In this study,effects of ethanol extract of Aquilaria sinensis leaves on hyperglycemia were investigated in diabetic db/db mice.After 4 weeks of administration,the 95%ethanol（EtOH） extract of Aquilaria sinensis leaves （AE）,especially at high dose level（600 mg/kg）,activated AMP-activated protein kinase（AMPK）,resulting in reduced fasting blood glucose and glycosylated hemoglobin levels in db/db mice.In addition,the oral glucose tolerance test（OGTT test） showed that AE could remarkably improve insulin resistance.Compared to Thiazolinediones（TZDs）,no weight gain was observed after AE administration,which is a severe side effect of TZDs.The data suggested that AE could act as an activator of AMPK,and might be used as an alternative to TZDs in the management of obesity-related diabetes.

Study on the regulatory effect of herbal cake-partitioned moxibustion on colonic CD206,AMPK and TSC2 in rats with Crohn disease

Objective:To explore the mechanism of herbal cake-partitioned moxibustion in Crohn disease(CD)treatment by observing the effect of herbal cake-partitioned moxibustion on protein expressions of colonic M2 macrophage marker CD206,AMP-activated protein kinase(AMPK)and tuberous sclerosis complex(TSC)2.Methods:Twenty-six specific pathogen free male rats were randomly divided into a normal group,a model group and a herbal cake-partitioned moxibustion group.The CD model was prepared by enema with the mixture of 5%(W/V)2,4,6-trinitrobenzene sulfonic acid(TNBS)and 50%ethanol at 2:1(volume ratio).After the model was successfully prepared,rats in the herbal cake-partitioned moxibustion group received herbal cake-partitioned moxibustion at Qihai(CV 6)and bilateral Tianshu(ST 25).Hematoxylin-eosin(HE)staining was used to observe the histopathological changes of rat colon;immunohistochemical technique was used to detect the expression of colonic CD206 protein;Western blot,immuno fluorescence,and real-time fluorescence quantitative polymerase chain reaction(RTqPCR)technologies were used to detect the protein and mRNA expressions of colonic AMPK and TSC2.Results:Compared with the normal group,rats in the model group showed damaged colonic mucosa,missing of the epithelial layer;thickened submucosa,vascular proliferation,massive infiltration of monocytes and lymphocytes,and cracked ulcers that reached the muscle layer.Rats in the herbal cake-partitioned moxibustion group showed reduced intestinal inflammation and healing intestinal epithelium ulcers.Compared with the normal group,rat colonic CD206 protein expression,and the protein and mRNA expressions of colonic AMPK and TSC2 were decreased in the model group(all P<0.Ol);compared with the model group,rat colonic CD206 protei n expression was in creased(P<Q.Q1),as well as the protein and mRNA expressions of AMPK and TSC2 in the herbal cake-partitioned moxibustion(all P<0.05).Conclusion:Herbal cake-partitioned moxibustion can reduce intestinal inflammation in CD rats,increase colonic CD206 protein expression,and up-regulate the protein and mRNA expressions of colonic AMPK and TSC2.

Study on the relationship between relieving energy crisis in myofascial trigger points with An-Pressing manipulation and AMPK/PGC-1α pathway activation

Objective To explore the mechanism of An-Pressing manipulation in relieving energy crisis in chronic myofascial trigger points(MTrPs)by observing the effects of An-Pressing manipulation on adenosine triphosphate(ATP),adenosine 5′-monophosphate(AMP)-activated protein kinase(AMPK)/peroxisome proliferator-activated receptorγcoactivator 1α(PGC-1α)pathway and mitochondrial ultrastructure of skeletal muscle cells in MTrPs rats.Methods Forty-eight male Sprague-Dawley rats were randomly divided into a blank group,a model group,a lidocaine group,and an An-Pressing manipulation group,with 12 rats in each group.The model group,lidocaine group and An-Pressing manipulation group were used to replicate the MTrPs rat model by blunt shock and centrifugal motion method.After modeling,the An-Pressing manipulation group was subjected to 7 times An-Pressing manipulation,once every other day;the lidocaine group was treated with 3 times of injection of lidocaine at the MTrPs,once every 6 d.The blank group and the model group were fed normally without intervention.After the intervention,local muscle tissue was taken to detect the content of ATP and the expression of AMPK,phosphorylated AMPK(phospho-AMPK),PGC-1α,and glucose transporter 4(GluT4),and the ultrastructure of mitochondria was observed under an electron microscope.Results Compared with the blank group,the ATP content in the model group was decreased(P<0.05),the protein expression levels of phospho-AMPK,PGC-1α,and GluT4 and the ratio of phospho-AMPK to AMPK were decreased(P<0.05);under the electron microscope,the number of mitochondria decreased,and they were deformed,small in volume,and had deformed cristae.Compared with the model group,the ATP contents in the An-Pressing manipulation group and the lidocaine group were increased(P<0.05),and the protein expression levels of phospho-AMPK,PGC-1α,and GluT4 and the ratio of phospho-AMPK to AMPK were increased(P<0.05);under the electron microscope,the number of mitochondria increased,the shape and size of the mitochondria were basically normal,and the cristae could be seen.Compared with the lidocaine group,phospho-AMPK and the ratio of phospho-AMPK to AMPK in the An-Pressing manipulation group were increased(P<0.05);under the electron microscope,the numbers of mitochondria were similar,and the shape and size of the mitochondria were basically normal without swelling,and the cristae could be observed.Conclusion An-Pressing manipulation can increase the ATP content in MTrPs tissue,improve the expression levels of PGC-1α and GluT4 proteins and the ratio of phospho-AMPK to AMPK;its mechanism may relate to the activation of AMPK/PGC-1α signaling pathway to promote the repair of mitochondrial damages.