Since the receptor for advanced glycation end products (RAGE)-ligand axis has been demonstrated to be important in fibrogenesis, rat models may be used to assess whether specific small interfering RNAs (siRNAs) that target RAGE are able to reduce the progression of hepatic fibrosis. However, the effect of RAGE-targeted siRNA on established hepatic fibrosis remains to be elucidated. In the present study, RAGE-specific siRNA expression vectors were constructed prior to the animal experiment. Sprague-Dawley rats were treated initially with olive oil (2 ml/kg) or 50% CCl4 (2 ml/kg; CCl4/olive oil=1:1) twice per week for six weeks to generate the fibrosis model. The rats were then treated with phosphate-buffered saline, a RAGE-specific siRNA expression vector, at different doses or a non-specific siRNA expression vector twice weekly via tail vein injection for up to six weeks, and were sacrificed at week two, four or six. Compared with the control groups, RAGE-specific siRNA therapy significantly decreased RAGE mRNA and protein expression in rat livers (P<0.01). Following six weeks of RAGE gene-silencing treatment, the liver function, which was assessed by analyzing serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL), improved to varying degrees (P<0.01). The expression of nuclear factor-κB (NF-κB) significantly decreased following RAGE gene-silencing therapy (P<0.01). In addition, the serum levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and extracellular matrix (ECM) components, including hyaluronic acid (HA), laminin (LN) and procollagen type III (PCIII) also decreased (P<0.01). Furthermore, the expression of α-smooth muscle actin (α-SMA) and collagen I, which indicate the activation of hepatic stellate cells (HSCs), were downregulated following RAGE gene-silencing therapy (P<0.01). Furthermore, the inflammatory activity grade and fibrosis stage of rat livers also significantly improved compared with the control groups following RAGE gene-silencing therapy. Specific targeting of RAGE using siRNA may inhibit RAGE gene expression effectively in the rat hepatic fibrosis model and attenuate the progression of established hepatic fibrosis. This therapeutic effect may be mediated via inhibition of the expression of NF-κB. These findings suggest that RAGE may be a new target to prevent hepatic fibrosis.
Liver cirrhosis, which is the end stage of several types of liver disease, is a global problem due to a lack of efficient therapies. Hepatic fibrosis is a necessary step in the development of liver cirrhosis. Therefore, the prevention and reversal of fibrosis is of high priority. Hepatic fibrosis is a pathological process that involves the deposition of extracellular matrix (ECM) (
The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super family of receptors (
RAGE is expressed in a broad range of cell types, including cardiomyocytes, vascular cells and inflammatory cells and, thus, is observed in HSCs (
Rat RAGE mRNA (GeneBank number, NM-053336.1) was used as the target sequence. RNA-designing software (
The
The vector pAKD.CMV.bGlobin.eGFP.H1.shRNA was first linearized by restriction enzyme digestion at the
RAGE siRNA expression vectors (pAKD-GR125, pAKD-GR126, pAKD-GR127, pAKD-GR128 and pAKD-GR129) were transfected into primary rat HSCs separately at multiplicities of infections (MOIs) of 20, 100, 200 and 1,000. Untreated and unspecific siRNA-transfected primary rat HSCs served as the controls. The medium was replaced with serum-free Dulbecco’s modified Eagle’s medium prior to transfection. Total RNA was extracted and the RAGE mRNA levels were determined using quantitative polymerase chain reaction (qPCR) following incubation for 48 h.
The specific siRNA expression vectors that resulted in maximum inhibition of RAGE gene expression were selected and transfected into primary rat HSCs and cultured for five days. Untreated and unspecific siRNA-transfected primary rat HSCs were used as the controls. The cells were harvested following incubation for 24, 48 or 72 h, respectively, and the total RNA was extracted. The efficiency of RAGE gene silencing was assessed using qPCR.
Six-week-old male Sprague-Dawley (SD) rats weighing 200±30 g were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). They were housed in the Animal Experiment Center of Medical College, Southeast University (Nanjing, China) under a 12-h dark/light cycle, and water and a standard diet were available
The study protocol was approved by the Animal Research Ethics Committee of Medical College, Southeast University (Nanjing, China).
Serum was collected from the blood samples by centrifuging at 10,000 × g for 8 min at 4°C. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) were measured using a Beckman LX20 autoanalyzer. The serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), hyaluronic acid (HA), procollagen type III (PCIII) and laminin (LN) were determined using radioimmunoassays, according to the methods of Barouch
Serial sections of liver tissue were prepared for pathological examination by staining with hematoxylin and eosin (H&E) and Masson. Two pathologists examined the slides independently under a light microscope. The severity of inflammation activity and fibrosis were graded based on the Knodell HAI evaluation system and Ishak-modified system (
Total RNA was extracted from rat liver samples using TRIzol reagent (Invitrogen, St. Louis, MO, USA) according to the manufacturer’s instructions. The RNA purity and concentration were determined and RNA was then reverse transcribed. qPCR was performed using a LightCycler PCR instrument with SYBR® green as the detection fluorophore (Applied Biosystems, Foster City, CA, USA). The thermal profile for PCR consisted of activation at 95°C for 3 min, followed by 40 cycles of PCR at 95°C for 20 sec, 60°C for 30 sec, and 72°C for 60 sec. Hepatic mRNA expression of type I collagen (collagen I), RAGE, α-smooth muscle actin (α-SMA) and NF-κB were evaluated using qPCR with β-actin used as the endogenous control. The ΔΔCT method was used to calculate relative differences in the expression levels of each target gene.
Sections of rapidly frozen liver tissue were generated using a freezing microtome and were observed under a fluorescence microscope. The amount of recombinant virus expression was assessed based on GFP expression.
Data are expressed as the mean ± standard deviation. Statistical analyses were performed with the SPSS statistical software package (version 17.0; SPSS, Inc., Chicago, IL, USA) using a one-way analysis of variance or independent-samples t-tests, as appropriate. P<0.05 was considered to indicate a statistically significant difference.
As compared with the untreated primary rat HSCs (blank) and the cells treated with pAKD-NS, the expression of RAGE mRNA was significantly downregulated in primary rat HSCs treated with pAKD-GR125 (P<0.05), pAKD-GR126 (P<0.05), pAKD-GR127 (P<0.05), pAKD-GR128 (P<0.05) and pAKD-GR129 (P<0.05). The levels of RAGE mRNA decreased in a dose-dependent manner over MOIs of 20–1,000. In particular, the largest decrease was observed following treatment with pAKD-GR126 at an MOI of 1,000 (
The GFP gene expressed by the pAKD-GR126 vector was used to trace siRNA expression. Whether the vector was transfected into liver tissues was judged from the expression of the GFP. Following sacrification, sections of rapidly frozen liver tissue were generated using a freezing microtome and were observed under a fluorescence microscope. Bright GFP fluorescence was observed in the liver tissues of therapeutic groups (LT, MT and HT group) and the NS group; however, not in the FM or NC groups (
A histological analysis of the rat liver sections was conducted, which were collected three days after the final injections, based on H&E and Masson staining. In the FM and NS groups, robust piecemeal or bridging necrosis, inflammatory-cell infiltration and connective tissue hyperplasia were observed in the portal area, and pseudo-lobular formation was also observed, whereas no hepatocellular necrosis, inflammatory cell infiltration or fibroplasia was observed in the NC group. Compared with the FM and NS groups, the severity of hepatic inflammation and fibroplasia was significantly decreased in the therapeutic groups (
The serum levels of ALT, AST, ALP and TBIL reflect liver function and hepatocyte injury in the liver. Compared with the therapeutic groups and the NC group, the levels of serum ALT, AST, ALP and TBIL in the FM group and the NS group were significantly higher (P<0.01;
HA, LN and PCIII are the main components of ECM and their serum levels may indirectly reflect the progression of hepatic fibrogenesis. The levels of serum HA, LN and PCIII were significantly lower in the therapeutic groups and the NC group (P<0.01;
RAGE mRNA expression in rat liver tissues was evaluated using qPCR. The mRNA expression of RAGE was low in normal rat liver tissues and it increased significantly following six weeks of CCl4 injections (P<0.01;
The mRNA expression of NF-
The expression of α-SMA is an important marker that may indicate the activation of HSCs. The mRNA expression of α-SMA in rat liver tissues was evaluated using qPCR. As compared with the NC group, CCl4 induced a strong upregulation of α-SMA mRNA, and RAGE-specific siRNA treatment significantly attenuated α-SMA mRNA expression. Following six weeks of RAGE gene-silencing therapy, when compared with the FM group, the relative expression of α-SMA mRNA in the LT, MT and HT group decreased by 4.92 (P<0.01), 21.86 (P<0.01) and 43.72% (P<0.01), respectively, and no significant difference in the mRNA expression of α-SMA was identified between the NS and FM groups (P>0.05;
The mRNA expression of collagen I in rat liver tissues was evaluated using qPCR. Collagen I is another important marker that indicates the activation of HSCs. Compared with the NC group, CCl4 injection markedly upregulated collagen I mRNA expression. Following RAGE-specific siRNA treatment for six weeks, the expression of collagen I mRNA significantly decreased in the LT group (28.83%; P<0.01), the MT group (43.24%; P<0.01) and the HT group (51.35%; P<0.01;
Following six weeks of RAGE gene-silencing therapy, rat livers were collected and the protein expression of RAGE, NF-κB, α-SMA and collagen I was evaluated using western blot analysis. The protein expression of these four markers demonstrated a similar trend to that of their respective mRNA expression profiles. The protein expression of RAGE, NF-κB, α-SMA and collagen I was significantly higher in the fibrosis model and was attenuated by RAGE gene-silencing therapy (P<0.01;
The present study reported, for the first time, to the best of our knowledge, the mechanisms of therapeutic effects of specific RAGE-targeting siRNA on CCl4-induced hepatic fibrosis in rats. The RAGE-ligand axis has been demonstrated to be important in fibrogenesis and a previous study by our group demonstrated that RAGE gene silencing effectively prevented liver fibrosis in a rat model (
In the present study, SD rats were used to generate a model of CCl4-induced hepatic fibrosis. Following six weeks of CCl4 injections, the liver-function biomarkers (ALT, AST, ALP and TBIL), the grade of hepatitis and the stage of hepatic fibrosis were much higher in the rats in the FM group than in those in the NC group, and the majority of rats in the FM group had ascites. These results indicated that the rat hepatic fibrosis model was established following six weeks of intraperitoneal injections with CCl4. Following interference with the RAGE-specific siRNAs, the rats in the therapeutic groups demonstrated improved liver functions compared with those in the FM and NS groups (P<0.01;
In addition, there is a growing body of evidence demonstrating that NF-κB may be important in HSC activation. The expression of α-SMA and collagen I, which reflect HSC activation, was also lower in the therapeutic groups when compared with the FM and NS groups (
Furthermore, NF-κB is a cardinal regulator of the inflammatory response by controlling the expression of genes encoding cytokines. The accumulation of pro-inflammatory cytokines provokes quiescent HSCs to undergo significant morphological and functional changes, together termed ‘activation’, and they transdifferentiate into proliferative, fibrogenic and contractile myofibroblast-like cells, which are considered to be the primary ECM-producing cells during pathogenic fibrosis (
In conclusion, the present study demonstrated that specific RAGE-targeted siRNA inhibited RAGE gene expression effectively in a CCl4-induced rat model of hepatic fibrosis and attenuated the progression of established hepatic fibrosis. Furthermore, the antifibrotic effect of RAGE gene silencing may be mediated by the inhibition of NF-κB expression, which mediates the inflammatory response and the activation of HSCs. These findings, together with a previous study by our group (
This study was funded by the Natural Science Foundation of Jiangsu Province, China (no. BK2009284).
Effect of different doses of RAGE-specific siRNA on the expression of RAGE mRNA in primary rat hepatic stellate cells. The expression of RAGE mRNA relative to β-actin mRNA was quantified using quantitative polymerase chain reaction. The changes were expressed as the percentage of the respective blank. *P<0.01 vs. blank and #P<0.01 vs. pAKD-NS. RAGE, receptor for advanced glycation end products; siRNA, small interfering RNA; MOI, multiplicity of infections; NC, normal control.
Effect of RAGE-specific siRNA on the expression of RAGE mRNA in primary rat HSCs at various time-points. The expression of RAGE mRNA relative to β-actin mRNA was quantified using quantitative polymerase chain reaction and the changes were expressed as the percentage of the respective blank.*P<0.01 vs. blank and #P<0.01 vs. pAKD-NS 48 h. RAGE, receptor for advanced glycation end products; HSCs, hepatic stellate cells; siRNA, small interfering RNA.
Expression of GFP in liver tissues. GFP fluorescence of liver tissues in the different groups was detected using a fluorescence microscope. (A) Fluorescence image of the liver tissue in the NC group; (B) fluorescence image of the liver tissue in the FM group; (C) fluorescence image of the liver tissue in the LT group; (D) fluorescence image of the liver tissue in the MT group; (E) fluorescence image of the liver tissue in the HT group, (F) fluorescence image of the liver tissue in the NS group. A-F, magnification, ×100. NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control; GFP, green fluorescent protein.
Histological examination of the effect of specific RAGE-targeted siRNA on rat hepatic fibrosis based on H&E and Masson staining. (A and B) NC; (C and D) FM; (E and F) LT; (G and H) MT; (I and J) HT; (K and L) NS. (A, C, E, G, I and K) H&E staining (magnification, ×40); (B, D, F, H, J and L) Masson staining (magnification, ×100). RAGE, receptor for advanced glycation end products; NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control; H&E, hematoxylin and eosin; siRNA, small interfering RNA.
Relative mRNA expression of RAGE in rat liver tissues of the different groups. *P<0.01 vs. NC; #P<0.01 vs. FM. RAGE, receptor for advanced glycation end products; FM, fibrosis model; NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control.
Relative mRNA expression of NF-κB in rat liver tissues of the different groups. *P<0.01 vs. NC; #P<0.01 vs. FM. FM, fibrosis model; NF-κB, nuclear factor-κB; NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control.
Relative mRNA expression of α-SMA in rat liver tissues of the different groups. *P<0.01 vs. NC; #P<0.01 vs. FM. FM, fibrosis model; α-SMA, α-smooth muscle actin; NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control.
Relative mRNA expression of collagen I in rat liver tissues of the different groups. *P<0.01 vs. NC; #P<0.01 vs. FM. FM, fibrosis model; NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control.
Effect of specific RAGE-targeted siRNA on the expression of 42-kD RAGE, 42-kD α-SMA, 50-kD NF-κB and 120-kD collagen I in rats. (A) Protein expression of 42-kD RAGE, 42-kD α-SMA, 50-kD NF-κB and 120-kD collagen I were determined using western blot analysis. (B) Amount of RAGE, α-SMA, NF-κB and collagen I was expressed relative to β-actin protein and was determined using densitometric scanning. The changes were expressed as the percentage of NC. *P<0.01 vs. NC; #P<0.01 vs. FM. RAGE, receptor for advanced glycation end products; α-SMA, α-smooth muscle actin; NF-κB, nuclear factor-κB; siRNA, small interfering RNA; NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control.
Effects of specific RAGE-targeted siRNA on rat liver tissue inflammation and fibrosis.
Group | n | Inflammation grade | Average rank | Fibrosis stage | Average rank | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
| ||||||||||||
0 | I | II | III | IV | 0 | I | II | III | IV | ||||
NC | 6 | 3 | 0 | 0 | 0 | 0 | 2.50 | 3 | 0 | 0 | 0 | 0 | 2.00 |
FM | 6 | 0 | 0 | 0 | 2 | 4 | 28.00 |
0 | 0 | 0 | 1 | 5 | 26.75 |
LT | 6 | 0 | 2 | 3 | 1 | 0 | 15.33 |
0 | 1 | 3 | 2 | 0 | 15.58 |
MT | 6 | 0 | 3 | 3 | 0 | 0 | 12.75 |
0 | 3 | 2 | 1 | 0 | 12.17 |
HT | 6 | 1 | 3 | 2 | 0 | 0 | 10.33 |
0 | 4 | 2 | 0 | 0 | 10.00 |
NS | 6 | 0 | 0 | 1 | 2 | 3 | 25.83 |
0 | 0 | 0 | 0 | 6 | 28.00 |
The rank sum test of the grade of inflammation in six groups, χ2=24.915, P<0.01. The rank sum test of the fibrosis stage in six groups, χ2=27.580, P<0.01.
P<0.01 vs. NC;
P<0.01 vs. FM.
RAGE, receptor for advanced glycation end products; NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control; siRNA, small interfering RNA.
Effect of specific RAGE-targeted siRNA on ALT, AST, ALP and TBIL levels in rats.
Time | NC | FM | LT | MT | HT | NS | |
---|---|---|---|---|---|---|---|
ALT (U/l) | Week 2 | 41.0±2.0 | 308.8±17.1 |
146.5±9.8 |
121.2±8.4 |
92.0±7.2 |
305.0±13.1 |
Week 4 | 41.0±3.6 | 286.7±11.3 |
108.0±8.3 |
91.3±7.6 |
68.3±6.7 |
288.5±9.7 | |
Week 6 | 42.7±2.5 | 270.8±13.2 |
94.7±9.5 |
74.7±6.7 |
50.8±5.5 |
279.3±8.5 | |
AST (U/l) | Week 2 | 96.7±4.7 | 396.2±10.2 |
197.2±6.8 |
181.3±8.8 |
176.0±6.1 |
405.3±9.4 |
Week 4 | 93.0±5.6 | 380.5±4.5 |
182.8±6.5 |
153.2±5.9 |
130.5±6.4 |
377.7±5.9 | |
Week 6 | 95.7±9.1 | 366.2±9.0 |
164.0±9.4 |
133.3±6.6 |
104.5±6.7 |
371.2±7.6 | |
ALP (U/l) | Week 2 | 88.0±4.6 | 291.0±7.8 |
223.3±4.8 |
205.8±6.2 |
189.8±8.2 |
291.8±6.4 |
Week 4 | 82.7±4.0 | 278.3±9.1 |
186.2±4.7 |
149.5±5.2 |
134.3±5.9 |
283.5±4.9 | |
Week 6 | 86.7±4.2 | 272.7±6.0 |
164.2±5.4 |
130.7±7.4 |
107.7±8.3 |
271.0±7.3 | |
TBIL (μmol/l) | Week 2 | 4.1±0.2 | 29.9±0.8 |
19.2±0.6 |
16.8±0.7 |
13.2±0.7 |
29.5±0.6 |
Week 4 | 4.2±0.1 | 27.8±0.7 |
16.8±0.6 |
12.8±0.6 |
8.6±0.6 |
27.9±0.8 | |
Week 6 | 4.1±0.2 | 25.5±0.9 |
15.4±0.8 |
10.1±0.7 |
6.6±0.9 |
24.3±0.6 |
P<0.01 vs. NC;
P<0.01 vs. FM.
RAGE, receptor for advanced glycation end products; NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TBIL, total bilirubin; siRNA, small interfering RNA.
Effect of RAGE-targeted siRNA on markers of fibrosis (HA, LN and PCIII) in rats.
Time | NC | FM | LT | MT | HT | NS | |
---|---|---|---|---|---|---|---|
HA (μg/l) | Week 2 | 111.3±6.1 | 431.7±6.3 |
325.7±5.2 |
280.0±6.1 |
244.7±6.3 |
418.0±10.0 |
Week 4 | 115.3±4.2 | 422.0±5.1 |
309.2±6.4 |
263.3±5.9 |
227.0±6.4 |
411.7±7.3 | |
Week 6 | 115.0±5.3 | 410.7±3.6 |
302.7±4.8 |
251.7±5.9 |
204.7±5.6 |
403.7±6.1 | |
LN (μg/l) | Week 2 | 84.7±5.7 | 231.0±6.7 |
216.7±6.2 |
206.5±4.1 |
194.5±6.2 |
230.5±8.4 |
Week 4 | 83.7±4.2 | 227.2±5.8 |
208.0±5.3 |
197.3±5.4 |
168.7±6.2 |
220.7±9.2 | |
Week 6 | 85.7±2.5 | 221.7±6.0 |
201.0±4.2 |
184.2±4.9 |
152.8±5.1 |
218.3±5.7 | |
PCIII (μg/l) | Week 2 | 108.0±2.0 | 366.0±7.4 |
310.5±5.4 |
275.0±6.3 |
239.2±4.4 |
374.7±5.3 |
Week 4 | 105.0±5.6 | 356.5±7.9 |
279.5±7.3 |
232.5±6.7 |
195.0±5.9 |
366.0±4.2 | |
Week 6 | 107.3±6.8 | 351.2±4.2 |
257.7±7.4 |
206.5±6.5 |
174.7±6.1 |
360.8±4.7 |
P<0.01 vs. NC;
P<0.01 vs. FM.
RAGE, receptor for advanced glycation end products; NC, normal control; FM, fibrosis model; LT, low-dose therapeutic; MT, medium-dose therapeutic; HT, high-dose therapeutic; NS, non-specific siRNA control; HA, hyaluronic acid; LN, laminin; PCIII, procollagen type III; siRNA, small interfering RNA.