Salamanders (which are lower vertebrates) are known to regenerate their amputated limbs. The “nAG” protein (nAG stands for newt Anterior Gradient) is the key protein mediating this form of regeneration [
The senior author (MMA) developed the original hypothesis that since “nAG” is a “regenerative” protein, it must also be an “antifibrotic” protein. Hence, a new nAG gene (suitable for higher vertebrates including humans) was designed, synthesized, and cloned. The cloned vector was successfully transfected into human fibroblasts. nAG expression was found to suppress the expression of collagen in human fibroblasts regardless of the presence of Transforming Growth Factor Beta (TGF
Liver fibrosis may be induced by several factors such as nonalcoholic fatty infiltration of the liver, alcoholic liver disease, viral hepatitis, autoimmune hepatitis, toxin-induced hepatitis, and hereditary metabolic diseases [
In the current study, we investigated the potential therapeutic properties of the nAG protein in liver fibrosis using a rat model.
This study was approved by our institutional review board and was conducted according to the Guidelines for Animal Experiments (Project# E-13-926).
We induced liver fibrosis in rats using carbon tetrachloride (CCL4) and this is a well-known model of rodent liver fibrosis [
At the end of the experiment, the serum level of six protein markers of liver fibrosis were measured in all rats using ELISA: hyaluronic acid, Platelet Derived Growth Factor-AB (PDGF-AB), Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), laminin, procollagen III N-terminal peptide (PIII-NP), and collagen type IV-Alpha 1 chain (collagen 4-
Furthermore, liver biopsies were taken from all rats at the end of the experiment (8 weeks) and the histological grading of liver fibrosis was graded using the Metavir scoring system [
Metavir histological grading of liver fibrosis.
Grade of fibrosis | Description |
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F0 | no fibrosis |
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F1 | Mild fibrosis: Fibrous portal expansion with mild localized fibrosis in the portal area |
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F2 | Moderate fibrosis: Portal fibrosis with few fibrous septa. |
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F3 | Severe fibrosis: Portal fibrosis with numerous fibrous septa. |
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F4 | Cirrhosis: Marked portal-to-portal and portal-to central fibrosis with regenerative nodules. |
Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 22.0 software (SPSS Inc., Chicago, Il, USA). For the study of serum protein levels, the means and Standard Deviations (SD) were calculated for the three groups (the control and the two experimental groups). We used the one-way analysis of variance (ANOVA) to compare the three groups and the post hoc test Dunnett T3 for multiple comparisons between the groups. P-value of < 0.05 was considered significant. For the histological grading of liver fibrosis, we compared the percentages of no/mild fibrosis versus moderate/severe fibrosis in the two experimental groups using the Fisher’s exact/Chi-square tests. P-value of < 0.05 was considered significant.
The results of the serum levels of 6 proteins in three groups are shown in Table
Results of the serum levels of various proteins in the three groups.
SERUM LEVEL OF: | Group I | Group II | Group III |
---|---|---|---|
(Control) | (CCL4 treatment) | (CCL4/nAG treatment) | |
N=15 | N=15 | N=15 | |
Hyaluronic acid | 15.133 ± 0.063 | 29.80 ± 6.145 | 15.739 ± 3.231 |
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PDGF-AB | 18.292 ± 0.023 | 202.839 ± 124.73 | 58.905 ± 29.198 |
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TIMP-1 | 35.563 ± 0.0299 | 449.25 ± 294.71 | 161.919 ± 67.518 |
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Laminin | 257.27 ± 12.91 | 1238.588 ± 622.419 | 415.34 ± 136.934 |
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Procollagen III N-terminal peptide | 0.242 ± 0.021 | 0.355 ± 0.050 | 0.161 ± 0.051 |
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Collagen Type IV- Alpha-1 chain | 2.933 ± 2.193 | 21.915 ± 9.226 | 3.08 ± 2.539 |
+There was a significant difference between the control group and CCL4/nAG treatment group since P < 0.05.
Hyaluronic acid doubled in the CCL4 group compared to the control group (P<0.05 between groups I & II) and the nAG treatment was able to normalize its serum levels with no significant difference between the control and the CCL4/nAG group.
The serum levels of PDGF-AB increased more than 10-fold in the CCL4 group compared to the control group (P<0.05 between groups I & II). The nAG treatment was able to significantly reduce (but not normalize) the serum levels of PDGF-AB. Hence, there was a significant difference (P<0.05) between groups I & III and also between groups II & III.
The serum levels of TIMP-1 increased more than 12-fold in the CCL4 group compared to the control group (P<0.05 between groups I & II). The nAG treatment was able to significantly reduce (but not normalize) the serum levels of TIMP-1. Hence, there was a significant difference (P< 0.05) between groups I & III and also between groups II & III.
The serum levels of laminin increased almost 5 folds in the CCL4 compared to the control group (P<0.05 between groups I & II). The nAG treatment was able to significantly reduce (but not normalize) the serum levels of laminin. Hence, there was a significant difference (P<0.05) between groups I & III and also between groups II &III.
The serum levels of PIII-NP were significantly increased in the CCL4 group compared to the control group (P<0.05 between groups I & II). The nAG treatment significantly reduced the serum level of PIII-NP below levels of the control group. Hence, there was a significant difference (P<0.05) between groups I & III and also between groups II & III.
Finally, the serum levels of collagen 4-
As expected, all rats in the control group (n=15) had no liver fibrosis and hence, these rats were not included in the statistical analysis of the grading of liver fibrosis. Table
The staging of liver fibrosis in the two experimental groups.
Degree of Fibrosis | Group II: CCL4 only | Group III: CCL4 and nAG |
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(n=15 rats) | (n=15 rats) | |
No or mild fibrosis | 2 (both rats had mild fibrosis) | 9 (3 rats had no fibrosis and 6 rats had mild fibrosis) |
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Moderate to severe fibrosis | 13 (4 rats had moderate fibrosis and 9 rats had severe fibrosis) | 6 (4 rats had moderate fibrosis and 2 rats had severe fibrosis) |
P= 0.008.
Our study is the first investigation of the therapeutic potential of the antifibrotic nAG protein in liver fibrosis. nAG treatment was able to significantly reduce the serum levels of several protein markers of liver fibrosis [
The pathophysiology of liver fibrosis is well described in the literature [
Our study also showed that the nAG treatment normalized the levels of hyaluronic acid (Table
The effectiveness of nAG in reducing the production of collagen production has been shown in human skin fibroblast [
The nAG treatment in our study was also able to normalize the serum levels of collagen 4-
Laminin in the liver is found both in the basement membrane (where it is associated with collagen type IV) as well as in the extracellular matrix (where it is associated with collagen types I & III) along the fibrous septa and within the space of Disse [
There are three important PDGF isoforms: PDGF-AA, PDGF-AB, and PDGF-BB. PDGF-AA selectively binds to PDGF-receptor alpha, while the latter two isoforms bind to both alpha and beta receptors of PDGF [
Many drugs have been proposed for the management of liver fibrosis [
nAG treatment was able to significantly reduce the serum levels of several protein markers of liver fibrosis and also significantly reduced the histological degree of liver fibrosis. Further studies are required to investigate the effect of nAG if combined with a carrier peptide to selectively increase its concentration in the stellate cells responsible for the fibrotic reaction in the liver.
Data are available at the College of Medicine Research Center, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia.
There are no conflicts of interest and no drug company is involved in funding.
This work was supported by the College of Medicine Research Center, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia.