The RAGE-specific and non-specific siRNA recombinant adeno-associated viral expression vectors (pAKD-GR126 and pAKD-NC, respectively) were constructed by the Research Center of Pharmaceutical Engineering and Process Chemistry, School of Pharmacy, East China University of Science and Technology (Shanghai, China).

Three healthy male, 8-week-old, clean-grade Sprague Dawley (SD) rats, weighing 400–500 g, were purchased from the Experimental Animal Center of Nanjing Medical University (license no. SCDK Su 2008-0004; Nanjing, China). Mouse desmin antibody and a streptavidin peroxidase kit were purchased from Zymed Life Technologies (Carlsbad, CA, USA) and used in the identification of HSCs. Following the separation and cultivation of primary rat HSCs, they were identified using inverted phase contrast microscopy (TS100; Nikon, Tokyo, Japan) to observe the refraction in the cell and the change of cell morphology in different training periods. It was observed that the newly separated live primitive HSCs did not stick to the wall, were spherical or round, bright, with a strong refractive index, and they contained more fat droplets in the cytoplasm. The primary HSCs were separated into the control, pAKD-GR126 and pAKD-NC groups after five days of culture. Prior to transfection, serum-free medium was used for the cell culture of each group. All groups received 200 mg/l AGE-bovine serum albumin (BioVision, Inc., Milpitas, CA, USA) for stimulation. The pAKD-GR126 group was subsequently directly transfected with the RAGE-specific siRNA expression vector pAKD-GR126, and the pAKD-NC group with the non-specific siRNA expression vector pAKD-NC.

In total, 108 male, 6-week-old SD rats, weighing 250–300 g, were purchased from the SIPPR-BK Laboratory Animal Co., Ltd. (license no. SCXK Hu 2008-0016; Shanghai, China). The rats were separated at random into the normal control (n=18) or model (n=90) groups. Rats in the normal control group were intraperitoneally injected with refined olive oil (2 ml/kg), twice a week for six weeks. Rats in the model group were intraperitoneally injected with 50% CCl₄ (2 ml/kg) mixed with olive oil at a ratio of 1:1, twice a week for six weeks, to establish the model of hepatic fibrosis. Once the model was established, the rats were separated at random into the fibrotic model (FM), pAKD-GR126 low-dose (LT), pAKD-GR126 medium-dose (MT), pAKD-GR126 high-dose (HT) and pAKD-NC (NS) groups, with 18 rats per group. The original normal control (NC) group was maintained as a blank control group. The NC and FM group rats were high-power injected with phosphate-buffered saline (2 ml/kg) via the tail vein with a 1-ml syringe, as this is a fast method of introducing material. Rats in the LT, MT and HT groups were high-power injected with recombinant virus pAKD-GR126 at a dose of 6×10¹⁰, 3×10¹¹ or 1×10¹² particles, respectively, via the tail vein. Rats in the NS group were high-power injected with recombinant virus pAKD-NC at a dose of 1×10¹² particles via the tail vein, twice a week for a total of six weeks. For all groups, blood was extracted from the heart chamber three days after the final tail vein injection, and the liver tissues were also sampled for detection. The present study was conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was approved by the Institutional Animal Care and Use Committee of Southeast University (Nanjing, China).

Type IV collagenase, pronase E and Nycodenz® were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol® reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). DNA enzyme I was purchased from Gibco Life Technologies (Carlsbad, CA, USA). The total RNA of each group was extracted using TRIzol according to the manufacturer's instructions, prior to the purity and concentration of the RNA being calculated: Samples of RNA (1.0 µl) with RNase-free diethyl pyrocarbonate water (1,000 µl) were blended together and the RNA concentration was measured using a GeneQuant spectrophotometer (GE Healthcare Life Sciences, Shanghai, China). The RNA was reverse transcribed into cDNA for the qPCR analysis. The qPCR protocol was as follows: 94°C for 3 min; 40 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; and finally 72°C for 5 min. The 2^−ΔΔCt method was used to calculate the relative inhibition rates. The gene primers for RAGE, TNF-α, IL-6 and β-actin were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), and the specific sequences are presented in Table I.

The HSC lysate of each group was collected to extract the proteins, and a bicinchoninic acid assay kit (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China) was used to detect the concentrations of the protein samples. The protein samples were then subjected to SDS-PAGE (10%), followed by transferal onto polyvinylidene difluoride membranes. The membranes were blocked with skimmed milk for 1 h, and then incubated overnight with a rabbit monoclonal primary antibody to RAGE (cat. no, EPR12206; Abcam, Cambridge, UK), which was diluted with Tris-buffered saline and Tween 20 according to the manufacturer's instructions, at 4°C. Following repeated washing, the membranes were incubated with a horseradish peroxidase-labeled secondary antibody (Beijing ComWin Biotech Co. Ltd., Beijing, China) at room temperature for 1 h and washed again. An electrochemiluminescence assay was then performed for sample visualization, and gel electrophoresis was performed for scanning and optical density analysis. β-actin was used as a control and the ratio of stripe density between experimental and control groups was determined.

Following the sacrifice of the rats, the livers were removed and rapidly frozen. Fluorescence microscopy was then used to observe the green fluorescent protein (GFP)-labeled pAKD-GR126 and pAKD-NC expression in the liver sections. The remaining liver tissues were fixed with 4% paraformaldehyde, paraffin-embedded and processed into sections, followed by hematoxylin and eosin (HE) and Masson's trichrome collagen staining. Two pathologists assessed the images under double-blind conditions, and performed inflammatory-activity grading and fibrosis staging of the liver samples using the Scheuer modified scoring system (12).

Rats were anesthetized with ether, and then subjected to cardiac chamber puncture to obtain sample blood, which was centrifuged at 12,683 × g for 8 min at 4°C. The upper part of the stratified serum was collected for the determination of TNF-α and IL-6 concentrations in the HF rats by radioimmunoassay, in accordance with the manufacturer's instructions. The TNF-α and IL-6 radioimmunoassay kits were obtained from the Naval Medical Research Institute (Shanghai, China).

SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA) was used for the statistical analysis. Measurement data met the normality and homogeneity of variance, and are expressed as the mean ± standard deviation. Multiple data were compared using analysis of variance, while pairwise comparisons used the Student-Newman-Keuls method. Grading group data used the rank sum test, with P<0.05 considered to indicate a statistically significant difference.

In the primary HSCs transfected with the RAGE-specific siRNA expression vector pAKD-GR126, the RAGE mRNA expression levels were reduced to 42.32±6.16 and 43.24±7.50% of the values in the control and pAKD-NC groups, respectively (F=7.791; P<0.05). The TNF-α mRNA expression levels were also reduced, to 38.68±4.11 and 39.50±3.29% of the values in the control and pAKD-NC groups, respectively (F=474.568; P<0.05). Similarly, the IL-6 mRNA expression levels were reduced to 47.46±5.52 and 45.94±4.55% of the values in the control and pAKD-NC groups, respectively (F=203.463; P<0.05). The differences between the pAKD-GR126 and control/pAKD-NC groups were statistically significant (P<0.05), while no statistically significant difference was found between the control and pAKD-NC groups (P>0.05). These results indicate that the RAGE-specific siRNA expressed by pAKD-GR126 was able to inhibit the mRNA expression of RAGE, TNF-α and IL-6 (Table II).

Western blot analysis results revealed that, following the transfection of the specific siRNA expression vector, the protein expression levels of RAGE, TNF-α and IL-6 in the HSCs were significantly reduced. RAGE protein expression levels were reduced to 51.06±13.79 and 47.94±5.36% of the values in the control and pAKD-NC groups, respectively (F=36.513; P<0.05); TNF-α protein expression levels were reduced to 57.00±6.07 and 56.01±5.27% of the values in the control and pAKD-NC groups, respectively (F=123.500; P<0.05); and IL-6 protein expression levels were reduced to 48.30±3.26 and 50.50±3.61% of the values in the control and pAKD-NC groups, respectively (F=320.555; P<0.05). No statistically significant difference was found between the control and pAKD-NC groups (P>0.05). These results indicate that the RAGE-specific siRNA expressed by pAKD-GR126 was able to inhibit the protein expression of RAGE, TNF-α and IL-6 (Table III and Fig. 1).

The expression vector pAKD-GR126 simultaneously expressed the specific siRNA and GFP; therefore, observation of GFP expression was used to determine the transfection efficiency of the expression vector, in addition to the expression of specific siRNA. Green fluorescence was observed in the LT, MT, HT and NS groups, while the NC and FM groups exhibited no fluorescence. The results therefore showed that the expression vector pAKD-GR126 was successfully transfected into the rat liver cells and was able to express the RAGE-specific siRNA (Fig. 2).

Compared with the NC group, the serum TNF-α and IL-6 levels of the FM group were significantly increased by 175.27±5.48 and 292.32±1.62%, respectively (P<0.05); however, after six weeks of treatment with RAGE-specific siRNA, the serum TNF-α levels of the LT, MT and HT groups were reduced to 76.68±1.83, 69.00±2.36 and 62.29±2.44%, respectively, of the level in the FM group (F=416.397; P<0.05), while the IL-6 levels were reduced to 81.23±1.38, 65.32±2.03 and 48.87±1.13%, respectively, of the level in the FM group (F=1,716.659; P<0.05). The serum TNF-α and IL-6 levels in the FM and NS groups exhibited no significant difference (P>0.05). These results indicate that the RAGE-specific siRNA was able to effectively inhibit the generation of TNF-α and IL-6 in the HF rats (Table IV).

The HE and Masson's trichrome staining of the liver portal areas of the FM and NS groups revealed numerous regions of spotty, flaky or bridge-like necrosis, inflammatory cell infiltration, fibrous connective tissue proliferation and the formation of false lobules. The NC group exhibited no liver tissue necrosis, inflammatory cell infiltration or fibrosis. Compared with the FM and NS groups, the inflammation and fibrotic proliferation in the LT, MT and HT groups showed various degrees of improvement, with the HT group exhibiting the most evident alleviation. The Scheuer modified scoring system was used for the liver inflammatory-activity grading and fibrosis staging of each group. The liver inflammatory activity grade and fibrosis stage of each treatment group was significantly reduced compared with that in the FM and NS groups (χ²=28.825 and 31.318, respectively; P<0.01). These results indicate that the RAGE-specific siRNA was able to effectively improve the degree of liver inflammation and fibrosis in the HF rats (Fig. 3 and Table V).