Objective:To investigate the effect of lanatoside C on autophagy in hepatocellular carcinoma Huh-7 cells,and the possible mechanism of action.Methods:After Huh-7 cells were treated with gradient concentration of lanatoside C for 24 h,the IC50 value of lanatoside C on Huh-7 cells was detected by CCK-8 assay,and the role of intervention of gradient concentration of lanatoside C in the proliferation and clone formation of Huh-7 cells was analyzed by CCK-8 assay and clone formation assay.Western blotting was used to detect the expression levels of autophagy markers in Huh-7 cells,such as microtubule-associated protein light chain 3(LC3)-II,LC3-Ⅰ,p62,Beclin1,vacuolar protein sorting 34(VPS30),VPS15 and autophagy-related protein 3(ATG3)in Huh-7 cells,and the changes of LC3-II/LC3-Ⅰ ratio were analyzed.The effects of lanatoside C on the formation of autophagic vesicles in Huh-7 cells were observed by laser confocal microscopy,and the effects of lanatoside C on the formation of autophagic vesicles in Huh-7 cells were confirmed by artificial intelligence image analysis.Finally,the effects of lanatoside C on the interaction between Beclin1,VPS34 and VPS15 on the formation of autophagic vesicles in Huh-7 cells were detected by co-immunoprecipitation.Results:The IC50 value of lanatoside C on Huh-7 cells at 24 h was(0.49±0.07)μg/mL.The results of the CCK-8 assay and clone formation assay suggested that lanatoside C had a significant inhibitory effect on the growth of Huh-7 cells in a concentration-dependent manner(both P<0.01).The results of Western blotting assay showed that the LC3-II/LC3-Ⅰ ratio was significantly increased with the increase in the concentration of lanatoside C(P<0.01),while the expression level of Beclin1 protein was up-regulated(P<0.05),and the expression level of p62 protein was significantly down-regulated(P<0.01).The results of laser confocal microscopy showed that lanatoside C could significantly induce autophagic vesicle formation in Huh-7 cells(P<0.01).The results of artificial intelligence image analysis confirmed this effect of lanatoside C(all P<0.01).The results of co-immunoprecipitation confirmed that lanatoside C was able to promote the binding of Beclin1 to VPS34 and VPS15 with each other(both P<0.01).Conclusion:Lanatoside C significantly inhibits the proliferation of Huh-7 cells and and induces Huh-7 cells autophagy,the mechanism of this effect may be related to up-regulating Beclin1 expression and promoting the combination of Beclin1 protein with VPS34 and VPS15 proteins.

Objective:To investigate the effect of lanatoside C on autophagy in hepatocellular carcinoma Huh-7 cells,and the possible mechanism of action.Methods:After Huh-7 cells were treated with gradient concentration of lanatoside C for 24 h,the IC50 value of lanatoside C on Huh-7 cells was detected by CCK-8 assay,and the role of intervention of gradient concentration of lanatoside C in the proliferation and clone formation of Huh-7 cells was analyzed by CCK-8 assay and clone formation assay.Western blotting was used to detect the expression levels of autophagy markers in Huh-7 cells,such as microtubule-associated protein light chain 3(LC3)-II,LC3-Ⅰ,p62,Beclin1,vacuolar protein sorting 34(VPS30),VPS15 and autophagy-related protein 3(ATG3)in Huh-7 cells,and the changes of LC3-II/LC3-Ⅰ ratio were analyzed.The effects of lanatoside C on the formation of autophagic vesicles in Huh-7 cells were observed by laser confocal microscopy,and the effects of lanatoside C on the formation of autophagic vesicles in Huh-7 cells were confirmed by artificial intelligence image analysis.Finally,the effects of lanatoside C on the interaction between Beclin1,VPS34 and VPS15 on the formation of autophagic vesicles in Huh-7 cells were detected by co-immunoprecipitation.Results:The IC50 value of lanatoside C on Huh-7 cells at 24 h was(0.49±0.07)μg/mL.The results of the CCK-8 assay and clone formation assay suggested that lanatoside C had a significant inhibitory effect on the growth of Huh-7 cells in a concentration-dependent manner(both P<0.01).The results of Western blotting assay showed that the LC3-II/LC3-Ⅰ ratio was significantly increased with the increase in the concentration of lanatoside C(P<0.01),while the expression level of Beclin1 protein was up-regulated(P<0.05),and the expression level of p62 protein was significantly down-regulated(P<0.01).The results of laser confocal microscopy showed that lanatoside C could significantly induce autophagic vesicle formation in Huh-7 cells(P<0.01).The results of artificial intelligence image analysis confirmed this effect of lanatoside C(all P<0.01).The results of co-immunoprecipitation confirmed that lanatoside C was able to promote the binding of Beclin1 to VPS34 and VPS15 with each other(both P<0.01).Conclusion:Lanatoside C significantly inhibits the proliferation of Huh-7 cells and and induces Huh-7 cells autophagy,the mechanism of this effect may be related to up-regulating Beclin1 expression and promoting the combination of Beclin1 protein with VPS34 and VPS15 proteins.

Objective: To observe the clinical efficacy and safety of coil embolization of septal branch in the treatment of obstructive hypertrophic cardiomyopathy (HOCM). Methods: Eighteen patients with HOCM hospitalized in our department from September 2014 to October 2016 were enrolled in this study. There were 12 males and 6 females in this cohort and the age of patients ranged from 22 to 64 years old. Left ventricular outflow tract gradient (LVOTG) was derived from echocardiographic apical five-chamber view at pre-operation and at 48 hours and 6 months post operation. 24-hour Holter ECG examination was performed to assess the ventricular tachycardia, atrial fibrillation, atrioventricular block at 3 days and 6 months after the interventional operation. Routine ECG and creatine kinase-MB (CK-MB) examination were performed at pre-operation, at 6, 24 and 48 hours post operation. Cardiac troponin T (cTnT) was detected at pre-operation, at 24, 48 hours and 6 days post operation. The clinical symptoms (including chest tightness, chest pain, shortness of breath, syncope) and NYHA classification were assessed at 1, 6 months after the operation by telephone follow-up or outpatient clinic visit. Results: The average preoperative LVOTG detected by cardiac catheter was 103.6 (92.0, 115.0) mmHg (1 mmHg=0.133 kPa) , and the average LVOTG significantly reduced to 44.3 (41.6, 47.2) mmHg immediately after operation (P<0.01). The average ventricular septal thickness at 48 hours (19.2±3.1) mm and 6 months (17.8±2.8) mm after operation tended to be lower than the preoperative ventricular septal thickness ((20.4±3.5) mm, P>0.05). The echocardiographic derived average LVOTG at 48 hours and 6 months after operation was 42.9 (41.1, 45.5) and 39.1 (37.5, 41.0) mmHg, which were significant lower than the preoperative average LVOTG (94.3 (88.5, 101.8) mmHg, both P<0.01). LVOTG at 6 months after operation was significantly lower than that at 48 hours after the operation (P<0.05). The NYHA classification at 6 months after operation was significantly improved compared to pre-operation NYHA classification (P<0.01). During and after the operation, there was no complete atrioventricular block and ventricular tachycardia, no patient developed anterior wall and inferior myocardial infarction. Only one patient experienced transient left bundle branch block. During the 6 months following-up, there was no death, syncope, chest pain, palpitations, shortness of breath, paroxysmal dyspnea and/or lower extremity edema, ventricular tachycardia, atrioventricular block and atrial fibrillation, complete atrioventricular block and ventricular arrhythmia. Conclusion The coil embolization of septal branch is effective and safe for the treatment of patients with HOCM.

Objective To evaluate the value of the neutrophil-lymphocyte ratio (NLR) in elderly type 2 diabetic patients (T2DM) with coronary heart disease (CHD).Methods We performed a retrospective observational study on 228 patients undergoing coronary angiography in Guizhou Provincial People's Hospital from April 2014 to July 2015.Patients were divided into three groups:the simple T2DM group (n=77),simple CHD group (n=72),and T2DM complicated with CHD group (n=79).Meanwhile,70 healthy elderly subjects served as the control group.The white blood cell count,high-sensitivity C-reactive protein (hs-CRP) and other clinical and laboratory parameters were collected,and NLR was calculated.Risk factors for CHD in T2DM patients were determined by logistic regression analysis.Multiple stepwise regression analysis was adopted to identify factors influencing NLR.Results The white blood cell count,neutrophil count,NLR and hs-CRP level in the simple T2DM,CHD,and T2DM+CHD groups were higher than in the control group [(7.48 1.81) 109/L,(7.72± 1.89) 109/L,(7.98±2.12) 109/L vs.(6.22± 1.61) 109/L;(4.49±1.38) 109/L,(4.88±1.56) 109/L,(5.35±1.40) 109/L vs.(3.52±0.84) 109/L;(2.84± 0.77),(3.07±0.79),(3.34±0.83) vs.(1.58±0.42);(2.92±0.65) mg/L,(3.20±0.86) mg/ L,(4.98±1.10) mg/L vs.(1.105±0.23) mg/L;respectively,P＜0.05 or P＜0.01].The lymphocyte count in the simple T2DM,CHD,and T2DM+CHD groups were lower than in the control group [(1.57±0.41) × 109/L,(1.58±0.40) × 109/L,(1.61±0.48) × 109/L vs.(2.22± 0.51) × 109/L,P＜0.05].NLR and hs-CRP levels in the T2DM+CHD group were higher than in the former two groups (all P＜0.05).Pearson correlation analysis showed that NLR was positively correlated with the Gensini score and hs-CRP level (r=0.7455 and 0.7792,both P＜0.01).Logistic regression analysis showed that NLR,hs-CRP levels and glycosylated hemoglobin A1c (HbA1c) were the risk factors for CHD in T2DM patients (OR=4.331,3.997 and 2.928,all P＜0.05).Multiple stepwise regression analysis showed that NLR was positively correlated with fasting plasma glucose,HbA1 c levels and systolic blood pressure (β' =0.3133,0.4720 and 0.3069,all P＜0.05).Conclusions NLR may be a valuable predictive factor for CHD in elderly T2DM patients.

AIM: To investigate the effect of inhibiting myosin light chain kinase ( MLCK) on endothelin-1 (ET-1) induced proliferation and apoptosis of rat pulmonary artery smooth muscle cells (PASMCs).METHODS: Rat PASMCs were cultured and stimulated with ET-1.The cells were randomly divided into control group , ET-1 group and ET-1+MLCK inhibitor group (ET-1+M).Western blotting, MTT assay, [3H]-TdR incorporation and flow cytometry were employed to test the expression of myosin light chain (MLC) and MLCK, cell proliferation, cell cycle and apoptotic rate of PASMCs ,respectively .The phosphorylation of MLC was determined by glycerol-PAGE coupled with Western blotting .RE-SULTS:Compared with control group , the protein expression of MLCK and MLC phosphorylation significantly enhanced af -ter ET-1 stimulation.ET-1 markedly induced the proliferation and decreased the percentage of apoptotic rate in the PASMCs.However, pretreatment with ML-7, a MLCK inhibitor, significantly reversed the above effects induced by ET-1. CONCLUSION:MLCK inhibitor effectively inhibits the ET-1-induced proliferation and the cell cycle progression .

Objective To evaluate the feasibility and safety of a delivery pathway for the performance of percutaneous left atrial appendage (LAA) occlusion in experimental canine models. Methods Transseptal puncture was performed via femoral vein approach under fluoroscopic and angiographic guidance in 12 experimental dogs. A pigtail catheter was advanced into the left atrium (LA), which was followed by LA angiography. The diameters of the neck of LAA were measured on LAA angiogram obtained in appropriate projection. After the delivery sheath was advanced along the wire into LA, a pigtail catheter was inserted into the ostium of the LAA and the sheath was then advanced over the pigtail into the LAA. LAA angiography was then performed through the delivery sheath to confirm the position of the delivery sheath. One hour after the procedure both electrocardiography (ECG) and transthoracic echocardiography (TTE) were carried out in five dogs to check the results, immediately after which the five dogs were sacrificed to macroscopically observe the damages of the puncture site of inter-atrial septum as well as inside the LA and LAA. One hour and 2 weeks after the procedure TTE was conducted in the remaining 7 dogs and these dogs were followed up for one month. Results One dog died of pericardial tamponade during the operation. In 8 dogs the LAA was clearly displayed in the projection position of right anterior oblique (RAO) 30°/cranial (CRA) 20°,while in 3 dogs the LAA was well visualized in the projection position of RAO 30° , and in one dog in the projection position of RAO 30°/caudal (CAU) 20°. The diameter of LAA neck was (13.6 ± 5.2) mm. The delivery sheath was safely advanced into the LAA along the pigtail catheter in all dogs, and no air embolism, thrombus or pericardial tamponade occurred. Hematoma at puncture point of groin occurred in 2 dogs, which was absorbed through pressure dressing. Macroscopic examination of the heart performed immediately after the operation showed that no bloody pericardial effusion was found, and mild hematoma at posterior wall of LA was seen in one dog and mild damage of the upper-margin intima of LAA was noted in 2 dogs. The mean fluoroscopy time was (10.1 ± 2.5) minutes and the mean operation time was (58 ± 12) minutes. TEE showed no pericardial effusion 2 weeks after the procedure. During the follow-up period of one month no sudden death, stroke or infection occurred. Conclusion This method of placing the delivery sheath into the LAA is clinically safe and effective, and it can reliably establish a pathway to advance the LAA occluder into LAA.

Objective To discuss the clinical efficacy and safety of improved wire- maintaining technique in performing transcatheter closure of ventricular septal defects. Methods During the period from June 2011 to June 2013 at Changhai Hospital, percutaneous transcatheter closure of ventricular septal defect with improved wire-maintaining technique was carried out in 62 patients. According to the manipulation used , the patients were divided into traditional wire-maintaining technique group (group A, n = 30) and improved wire- maintaining technique group (group B, n = 32). The use of occluder during the procedure, the fluoroscopy time, the operation time and the complications were recorded. Follow-up examinations with ECG, echocardiogram and chest radiograph were performed at 24 hours and at 1 , 3 and 6 months after the procedure. The results were analyzed. Results No statistically significant differences in the use of occluder and in the incidence of complications existed between the two groups (P > 0.05). No severe complications occurred in both groups. The fluoroscopy time and the operation time in group A were (11.96 ± 3.63) min and (53.43 ± 14.48) min respectively, while the fluoroscopy time and the operation time in group B were (9.37 ± 2.77) min and (45.34 ± 10.38) min respectively, and the differences between the two groups were statistically significant (P < 0.05). Conclusion In performing transcatheter closure of ventricular septal defects, the practice.

Objective To explore the feasibility and effectiveness of interventional transcatheter destruction of the aortic valve to establish an animal model with acute aortic valve regurgitation. Methods Eight healthy goats were used for this study. A limited sternotomy approach was used to access the apex of the heart. Puncturing of the apex of the heart was performed to establish a wire track, then, under fluoroscopic guidance a 10 F sheath was inserted along this track of hard wire until to the ascending aorta above the aortic valve. The internal sheath was removed. Via the 10 F sheath a 10 mm occluder of ventricular septal defect (VSD) was introduced into the ascending aorta above the aortic valve. The sheath was pulled back to the left ventricle, while the occluder remained in the ascending aorta above the aortic valve. Then the occluder was quickly pulled back into the left ventricle in order to make some certain damage to the aortic valve. And an acute aortic valve regurgitation model was thus established. Angiography of ascending aorta above the aortic Among the 8 animals, two died of acute left ventricular failure on the spot due to excessive regurgitation blood flow after the operation. Macroscopically, damage of the aortic valve was seen. In the six survivors, angiography of ascending aorta above the aortic valve and Doppler echocardiography showed that moderate degree of regurgitation was detected in 5 and small amount of regurgitation in one. Two experimental goats with moderate degree of regurgitation died of heart failure separately at seven days and fifteen days after the operation. The remaining four experimental goats survived for more than three months. Follow- up checkups with echocardiography suggested the presence of mild- moderate degree of regurgitation. Conclusion Acute aortic valve regurgitation model in experimental goats can be established through transapical transcatheter damage of aortic valve by quickly pulling back a VSD occluder which has been placed in the ascending aorta above the aortic valve. This method is clinically feasible, technically simple and repeatable, the result is reliable, and the degree of regurgitation is controllable.

Objective To explore the effect of small dose of fentanyl combined with midazo-lam on prevention of radial artery spasm.Methods 1086 patients with elective transradial percuta-neous coronary intervention were randomly divided into medicine group and control group.The medicine group was treated with conventional intravenous injection of fentanyl and midazolam.Inci-dence rate of radial spasm,vascular complications and mortality rate or re-hospitalization rate with-in 30 days were recorded and compared between two groups.Results Incidence rate of radial spasm in medicine group was 2.5%,which was significantly lower than 8.8% in the control group (P <0.001).There were no significant differences in aspects of VAS score,mortality rate and re-hos-pitalization rate between two groups.Multivariate analysis showed that there were obvious correla-tion between incidence of radial spasm and gender,height,obesity,smoking,numbers of tubes, surgical way and difficulty of operation.Conclusion Small dose of fentanyl combined with midazo-lam during transradial interventional procedures can significantly reduce the incidence of radial spasm and vascular complications,and it won’t cause increasing of mortality rate and re-hospitalization rate.

Objective To explore the effect of small dose of fentanyl combined with midazo-lam on prevention of radial artery spasm.Methods 1086 patients with elective transradial percuta-neous coronary intervention were randomly divided into medicine group and control group.The medicine group was treated with conventional intravenous injection of fentanyl and midazolam.Inci-dence rate of radial spasm,vascular complications and mortality rate or re-hospitalization rate with-in 30 days were recorded and compared between two groups.Results Incidence rate of radial spasm in medicine group was 2.5%,which was significantly lower than 8.8% in the control group (P <0.001).There were no significant differences in aspects of VAS score,mortality rate and re-hos-pitalization rate between two groups.Multivariate analysis showed that there were obvious correla-tion between incidence of radial spasm and gender,height,obesity,smoking,numbers of tubes, surgical way and difficulty of operation.Conclusion Small dose of fentanyl combined with midazo-lam during transradial interventional procedures can significantly reduce the incidence of radial spasm and vascular complications,and it won’t cause increasing of mortality rate and re-hospitalization rate.