| BackgroundVentricular remodeling (VR) is the process activation of fetal gene program and the complex molecular signals change after heart damage and overload state cause by acute myocardial infarction (AMI). Cardiac hypertrophy is the major clinical manifestations of Ventricular remodeling. VR can lead to heart failure, sudden death. VR is one of the most important risk factor for long-term cardiac death. Lectin and-like oxidized LDL receptor(LOX-1) is a type II membrane protein of C-type hemagglutinin family, can be induced by a variety of pathological stimuli. Kai-Xin-Jiao-Nang (KXJN) can reduce cardiac hypertrophy, cardiac myocyte apoptosis, collagen remodeling in non-infarcted area In this study, we explored the role of LOX-1and the regulation mechanism of KXJN in ventricular remodeling after myocardial infarction by RNA interference, lentivirus, Western-blot and RT-PCR technology, for reconstruction therapeutic targets and effective drugs, rich of the mechanism of the traditional Chinese medicine treatment for the prevention of ventricular remodeling after myocardial infarction to explore a new ideas and approaches.Methods1The effect of KXJN on the LOX-1expression in cardiomyocyte hypertrophy with AMI We ligated the left main coronary artery to establish the AMI model. The SPF male Wistar rats which were conformed to the VR criteria were randomly divided into5groups (n=12, each):model group, KXJN group, captopril group; with sham group, a total of6groups. The rats were sacrificed after12weeks. HE staining was used to observe the myocardial hypertrophy, and western blot was used to detect the LOX-1expression level of the myocardial tissue.2The establishment and identification of the targeting LOX-1RNA interference lentiviral vectorConstruct LOX-1RNA interference lentivirus by pLL3.7lentivirus system3The establishment and identification of the over-expression LOX-1lentiviral vector.Construct overexpress LOX-1lentivirus by pHIV-H2BmRFP lentivirus system4. The effect of different concentrations of KXJN on H9C2myocardial cell viabilityAfter cultured for24h, drugs were prepared with DMEM medium containing1%fetal bovine serum. The cells were randomly divided into six groups:control group (control), KXJN2.5mg/ml group, KXJN5mg/ml group, KXJN10mg/ml group, KXJN20mg/ml group, KXJN40mg/ml group, KXJN80mg/ml group, six holes in each group. After cultured for24h, the MTT assay was used to observe cell activity.5.The effect of KXJN on Ang II-induced myocardial hypertrophy and the expression of LOX-1.After the control virus LV.NC infection of myocardial cells (H9C2NC cells) cultured for24h, drugs were prepared with DMEM medium containing1%fetal bovine serum, cells were divided into six groups:Control group, Ang Ⅱ group: AngⅡ10-7M, KXJN low-dose group (KXJN2.5mg/ml):Ang Ⅱ10-7M+KXJN2.5mg/ml; KXJN moderate dose group (KXJN5mg/ml):Ang Ⅱ10-7M+KXJN 5mg/ml; KXJN high dose group (KXJN10mg/ml):Ang II10-7M+KXJN10mg/ml, LOX-1RNAi group:Ang II of10-7M+H9C2LOX-1-RNAi cell. After cultured for24hours, immunofluorescence staining was used to observe the cell area, BCA assay was used to detect the cell protein content and the Westren bolt was used to detect the rat myocardial LOX-1expression levels.6. The effect of KXJN on H9C2LOX1+myocardial hypertrophyAfter H9C2NC and H9C2LOX-1+cells cultured for24h, drugs were prepared with DMEM medium containing1%fetal bovine serum. The cells were randomly divided into five groups:Control group (H9C2NC), over-expression of LOX-1group (LOX+): H9C2LOX-1+cells,KXJN low dose group (KXJN2.5mg/ml):H9C2LOX-1+cells+KXJN2.5mg/ml, KXJN moderate dose group (KXJN5mg/ml):H9C2LOX-1+cells+KXJN5mg/ml), Kaixin high dose group (KXJN10mg/ml):H9C2LOX-1+cell+KXJN10mg/ml). After cultured for24hours, immunofluorescence staining was used to detect the cell area, BCA assay was used to detect the cell protein content, the Westren bolt was used to detect the rat myocardial LOX-1expression levels.Result1. The effect of KXJN on cardiomyocyte hypertrophy of VR in ratsThe sizes of cardiomyocytes in different groups were significant difference (F=24.244, P=0.000). Compared with the control group, the cardiomyocytes size of the model group significantly increased (P=0.000). The cardiomyocytes size of Different doses of KXJN group and the captopril group were significantly smaller than that of control the model group (P=0.000). There were no significant differences of the cardiomyocytes size between the KXJN High does group and captopril group (P=0.707).2. The effect of KXJN on the expression of LOX-1with AMIThe myocardial LOX-1expression levels of different groups were significant different (F=44.281, P=0.000). Compared with the control group, the LOX-1expression level of the model group was obviously increased (P=0.000). The LOX-1expression level of KXJN group was lower than that of the model group, the difference was statistically significant (P=0.000). Compared with model group, the LOX-1expression levels of the captopril group, was significantly decreased (P=0.001). The LOX-1expression level of KXJN low does group is lower than that of the captopril group(P=0.000).3Construction of targeting LOX-1RNA interference lentiviral vectorafter72h The efficiency of Lentiviral infection is approximately95%. Different targets lentiviral infection H9C2cells for72h, the LV.LOX1-562group and LV.LOX1-628group LOX-1mRNA level decreased, compared LV.NC group difference was significant (LV.LOX1-562group:P=0.001; LV.LOX1-628:P=0.003). LV.LOX1-601LOX-1mRNA levels has declining trend compared with LV.NC, but the difference was no significant (P=0.157).4Construction of overexpression of LOX-1lentiviral vectorThe infection efficiency is approximately95%. Compared with negative lentiviral infection group, LOX-1mRNA levels was increased significantly (t=-18.68, P=0.000).5The effect of different concentrations of KXJN on H9C2myocardial cell viabilityThe effects at different concentrations of KXJN water extracts on myocardial cells were were significant different (F=535.588, P=0.000). Compared with the Control group, the cell activities were no significant difference between the low doses of the drug concentration (5-20mg/ml)(P=0.842, P=0.062, P=212). Compared with the Control group, the cell viability of40mg/ml for24hours significant decreased by approximately30%(P=0.000). Compared with the Control group, the cell viability of60mg/ml for24hours obviously decreased by60%(P=0.000). Compared with the Control group, cell viability of80mg/ml for24hours significantly decreased by80%(P=0.000). Accordingly, the KXJN aqueous extract concentration of2.5,5,10mg/ml were used in this study to exclude the drug itself toxic effects on cells.6. The effect of KXJN on Ang II-induced myocardial hypertrophyThe cardiomyocytes size of different groups were significant difference (F=F=126.041, P=0.000). Compared with Control group, cardiomyocytes size of Ang II group increased by a significant difference (P=0.000). Different doses of KXJN group and the LOX-1RNAi group, myocardial cell sizes were significantly less than the Ang II group(P=0.000). The cardiomyocytes sizes of KXJN moderate dose and high dose group were less than the low dose group, the significant difference was compared with the low dose group (KXJN moderate dose group:P=0.014; Kaixin high dose group:P=0.000). The cell size of high dose group of KXJN was less than the moderate dose group, compared with the moderate dose group, the difference was statistically significant (P=0.000). Compared with the LOX-1RNAi group, the cardiomyocytes sizes of KXJN moderate, low-dose group were significantly larger than the LOX-1RNAi group (P=0.000). Compared with the LOX-1RNAi group, the myocardial cell size of KXJN high dose group was less than the LOX-1RNAi group, but the difference was no statistically significant (P=0.271).The myocardial cell protein content of different groups have significant differences (F=36.121, P=0.000). Compared with the Control group, myocardial cell protein content of Ang II group significantly increased (P=0.000). The myocardial protein content of different doses of KXJN group and the LOX-1RNAi group, were lower than the Ang II group, and the difference was statistically significant (KXJN low dose group:P=0.004; other groups:P=0.000). Compared with the low dose group, the myocardial protein contents of KXJN moderate dose and high dose group were significantly lower than the low dose group (KXJN moderate dose group:P=0.009; KXJN high dose group:P=0.000). compared with the moderate dose group, the protein content of KXJN high dose group was lower than the moderate dose group, and the difference was statistically significant (P=0.014). Compared with the LOX-1RNAi group, the myocardial protein contents of KXJN moderate dose and low dose group were lower than the losartan group, and the difference was statistically significant (KXJN low dose group:P=0.000; KXJN moderate dose group:P=0.001). KXJN high dose group cardiomyocytes size was lower than the LOX-1RNAi group, but the difference has no statistically significant (P=0.313).7The effect of KXJN on Ang II-induced LOX-1expressionThe myocardial LOX-1expression levels of different groups were significant different (F=83.259, P=0.000). Compared with the control group, the LOX-1expression level of the Ang II group was obviously increased (P=0.000). The LOX-1expression levels of different doses of KXJN group and the LOX-1RNAi group, were lower than the Ang II group, and the difference was statistically significant (P=0.000). Compared with the low dose group, the LOX-1expression levels of KXJN moderate dose and high dose group were significantly lower than the low dose group (P=0.000). Compared with the moderate dose group, the LOX-1expression levels of KXJN high dose group was lower than the moderate dose group, and the difference was statistically significant (P=0.000). Compared with the LOX-1RNAi group, the LOX-1expression levels of KXJN moderate dose, low dose group and high dose group were lower than the LOX-1RNAi group, and the difference was statistically significant (KXJN high dose group:P=0.006; KXJN moderate dose and low dose group:P=0.000).8The effect of KXJN on cardiac myocyte hypertrophy of H9C2LOX-1+cardiac cellsThe cardiomyocytes size of different groups were significant difference (F=343.583, P=0.000). Compared with Control group, cardiomyocytes size of LOX+group increased by a significant difference (P=0.000). Myocardial cell sizes of different doses of KXJN group were significantly less than the LOX+group(P=0.000). The cardiomyocytes sizes of KXJN moderate dose and high dose group were less than the low dose group, the significant difference was compared with the low dose group (P=0.000). The cell size of high dose group of KXJN was less than the moderate dose group, compared with the moderate dose group, the difference was statistically significant (P=0.000).The myocardial cell protein content of different groups have significant differences (F=209.101, P=0.000). Compared with the Control group, myocardial cell protein content of LOX+group significantly increased (P=0.000). The myocardial protein content of different doses of KXJN group were lower than the LOX+group, and the difference was statistically significant (P=0.000). Compared with the low dose group, the myocardial protein contents of KXJN moderate dose and high dose group were significantly lower than the low dose group (P=0.000). Compared with the moderate dose group, the protein content of KXJN high dose group was lower than the moderate dose group, and the difference was statistically significant (P=0.000).9The effect of KXJN on LOX-1expression of H9C2LOX-1+cardiac cellsThe LOX-1expression of different groups were significant difference (F=F=308.125, P=0.000). Compared with Control group, LOX-1expression of LOX-1+group increased by a significant difference (P=0.000). LOX-1expression of different doses of KXJN group were significantly less than the LOX-1+group(P=0.000). The LOX-1expression of KXJN moderate dose and high dose group were less than the low dose group, the significant difference was compared with the low dose group (P=0.000). The LOX-1expression of high dose group of KXJN was less than the moderate dose group, compared with the moderate dose group, the difference was statistically significant (P=0.000).Conclusion1KXJN is a effective drug for the treatment of ventricular remodeling2KXJN inhibit cardiac hypertrophy through LOX-1signaling pathway3LOX-1play a key role in cardiac hypertrophy4562fragments of LOX-1gene is the ideal sites for RNA interference... |
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