ABSTRACT
Objective To investigate the effects of HMGB1 small interference RNA (siRNA) on retinal vascular endothelial cells.Methods siRNA was used to inhibit the expression of HMGB1,followed by the application of CCK8 assay,Hochest33342 staining and flow cytometry to observe the effects of HMGB1 siRNA on retinal vascular endothelial cells in high glucose environment.Meanwhile,the expression of proteins related to apoptosis was detected by Western blot.Results The transfection of HMGB1 siRNA down-regulated the protein expression level of HMGB1 by 73％ in siRNA group compared with normal control (NC) group (P ＜ 0.05),and the protein expression level of HMGB1 in siRNA group was decreased by 75％ compared with scr-siRNA group (P ＜ 0.05).There was no significant difference between NC group and scrsiRNA group (P ＞ 0.05).The total apoptotic rate of NC group,high-glucose group,scrsiRNA group and siRNA group was (0.40 ± 0.03)％,(49.80 ± 3.50)％,(47.60 ±1.98) ％ and (23.60 ± 2.40) ％ by flow cytometry.Compared with NC group,the apoptotic rates of high-glucose group,scr-siRNA group and siRNA group were increased (all P ＜ 0.05).Compared with scr-siRNA group,the apoptotic rate of HRECs in siRNA group was reduced,with significant statistical difference (P ＜ 0.05).There was no significant difference in the rate of cell apoptosis between scr-siRNA group and high-glucose group (P ＞ 0.05).Compared with the NC group,the protein expression level of cleavedcaspase3 protein in high-glucose group and scr-siRNA group were increased by (233 ±10) ％ and (266 ± 22) ％,respectively (both P ＜ 0.05);compared with scr-siRNA group,the protein expression level of cleaved-caspase 3 in siRNA group was reduced by (43 ±3) ％ (P ＜ 0.05);and there was no significant difference in the protein expression of cleaved-caspase 3 in high-glucose group and scr-siRNA group (P ＞ 0.05).Compared with the NC group,the protein expression of B-cell lymphoma-2 (Bcl-2) in high-glucose group and scr-siRNA group was decreased by (32 ± 2) ％ and (29 ± 3) ％,accordingly (both P ＜ 0.05);compared with scr-siRNA group,the protein expression level of Bcl-2 in siRNA group was increased by (42 ± 2) ％ (P ＜ 0.05);and there was no significant difference in the protein expression of Bcl-2 in high-glucose group and scr-siRNA group (P ＞ 0.05).Conclusion HMGB1 siRNA can reduce the apoptosis of retinal vascular endothelial cells in high glucose environment by inhibiting the activation of cleavedcaspase3 and increasing the expression of Bcl-2.
ABSTRACT
<p><b>AIM</b>To establish a simple, rapid and accurate electroanalytical method for water soluble porphyrin meso-tetrakis-(4-sulfonatophenyl) porphyrin (TPPS4); to clarify the reaction between water soluble porphyrins and bovine serum albumin (BSA); and to determine the interaction of TPPS4 with BSA in the absence of presence of cyclodextrins (CDs), separately.</p><p><b>METHODS</b>Three methods including LSV, UV spectroscopy and fluorescence spectroscopy had been employed to the relevant experiments. The way of employing three methods at the same time could make the experiment results more reliable.</p><p><b>RESULTS</b>In the supporting electrolyte of NaH2 PO4-Na2 HPO4 (pH 7.18), a sensitive reduction peak of TPPS4 was found by linear sweep voltammetry (LSV), the peak potential (Ep) was -0.70 V (vs SCE). The relationship between the second derivative peak of LSV (ip") and the concentration of TPPS4 was linear from 1.0 x 10(-7) mol x L(-1) to 1.0 x 10(-5) mol x L(-1), the square of correlation coefficients (r2) were 0.998 3 and 0.999 3, respectively. The relative standard deviation (RSD) was 0.56% (n = 5). The mean recovery of TPPS4 was 99.59%. In NH4Cl-NH3 x H2O buffers (pH 9.05), it was proved that BSA and TPPS4 could interact with each other and form 1 : 1 TPPS4-BSA supramolecular system. Moreover, the interaction between TPPS4 and BSA had been investigated by adding cyclodextrins (CDs). The interaction of TPPS4 with BSA was facilitated both by hydroxypropyl-beta-CD (HP-beta-CD) and sulforbutylether-beta-CD (SBE-beta-CD).</p><p><b>CONCLUSION</b>An electroanalytical method for TPPS4 has been established by LSV. The porphyrin drugs included by CDs could react with protein existing inside the human body easier. The consequences of this article also show that CDs will play important role in controlling and releasing the porphyrin drugs.</p>