Effects of dengue type 2 virus on the apoptosis and autophagy of primary HHSECs and the expression of related genes
Objective 
To investigate the effects of dengue type 2 virus (DENV-2) on the apoptosis and autophagy of primary human hepatic sinusoidal endothelial cells (HHSECs) and the expression of ICAM-1 and Beclin-1 at mRNA level and to analyze the possible pathogenic mechanism of DENV-2. 
 
 
Methods 
Immunohistochemistry (IHC) and flow cytometry analysis (FCM) were performed to identify HHSECs by detecting factor Ⅷ and CD31. The DENV-2 strain was identified by using PCR and HindⅢ. The 50% tissue culture infective dose (TCID50) of DENV-2 was calculated after infecting C6/36 cells with DENV-2. Dynamic changes of DENV-2 NS1 were measured by real-time PCR after infecting HHSECs with DENV-2. CCK-8 was used to dynamically detect the cytotoxicity of DENV-2 to HHSECs. The transcriptional levers of Beclin-1 and ICAM-1 in DENV-2-infected HHSECs were detected by real-time PCR. FCM was performed to analyze the apoptosis of HHSECs and the expression of LC3B and ICAM-1. 
 
 
Results 
The cells in the experimental group were stained brown by DAB and the positive expression rate of CD31 reached 87.1%. The TICD50 of DENV-2 to C6/36 cells was 10-6.845/0.1 ml. Compared with the uninfected cells, partial sequences of NS1 gene were expressed in DENV-2-infected HHSECs. DENV-2 suppressed the cell activities of HHSECs. The suppression rates of DENV-2 to HHSECs at 12 h, 24 h, 36 h and 48 h were respectively (10.90±1.24)%, (16.40±0.42)%, (17.00±0.46)% and (29.60±0.26)% (P<0.05). The transcriptional levels of Beclin-1 and ICAM-1 in HHSECs were significantly increased at the time point of 24 h after DENV-2 infection, the 2-△△Ct values of which were 46.77±2.55 and 40.97±4.91, respectively. The expression of LC3B and ICAM-1 in DENV-2-infected HHSECs were increased, the peaks of which were reached at 24 h (14.7%) and 36 h (35.5%), respectively. The apoptosis of DENV-2-infected HHSECs was remarkably enhanced at 12 h with an apoptosis rate of 13.17%. 
 
 
Conclusion 
HHSECs was susceptible to DENV-2. DENV-2 induced the upregulation of ICAM-1 and the activation of HHSECs. Moreover, autophagy and apoptosis of HHSECs could also be induced by DENV-2. 
 
 
Key words: 
Dengue virus; Primary HHSEC; Apoptosis; Autophagy; Beclin-1; ICAM-1