Encouraging effects of Viusid on liver histology have been reported in patients with chronic hepatitis C and nonalcoholic fatty liver disease[27,31
]; however, pathophysiologic mechanisms for explaining these effects remain unknown. There is a potential hepatoprotective mechanism in the chemical composition of Viusid that could be explained by the anti-inflammatory and antioxidant properties of its different molecules, such as zinc, glycyrrhizin acid, and ascorbic acid[24-26
]. Thus, the aim of this study was to investigate the role of Viusid as an antioxidant and an immunomodulator in patients with CHC who have failed previous antiviral treatment.
Several studies have demonstrated increased production of MDA in CHC, which is an indirect marker for oxidative stress (lipid peroxidation), and its levels have been associated with moderate-to-severe inflammation and fibrosis compared with those patients who had milder disease, suggesting higher oxidative stress with advanced stage of the disease[32,33
We found that MDA and 4-hydroxyalkenal levels were significantly reduced in the serum of the patients treated with Viusid as compared with placebo, indicating an important effect on lipid peroxidation products. Recent evidence shows that increased lipid peroxidation products, such as 4-hydroxynonenal, play a decisive role in stellate cell activation and fibrosis progression[34
]. Increased 4-hydroxynonenal levels upregulate procollagen and tissue inhibitor of metalloproteinase-1 gene expression. Matrix metalloproteinase-1 plays an important role in degrading collagen; therefore, the inhibition of this enzyme could increase fibrosis. Thus, this provides a putative mechanism to explain in part the effect of Viusid on fibrosis reduction in patients with CHC[27
]. Therefore, these results support the rationale for further studies in other forms of liver disease such as alcoholic liver disease and nonalcoholic fatty liver disease.
Encouraging antioxidant effects of ascorbic acid and zinc have been reported recently. Ascorbic acid and zinc supplementation have been associated with a significant reduction of LPO products[35,36
Surprisingly, a marked reduction of serum antioxidant defense system markers was seen in the whole group during treatment, maybe reflecting the “regression towards the mean” phenomenon, a placebo-induced effect, or poor maintenance of the redox homeostasis status.
Imbalanced Th1- and Th2-type response has been postulated to play an important role in influencing both the persistence of HCV infection and the extent of liver damage[10,37,38
]. In this context, IFN-γ and IL-12 are Th1-type cytokines, which are critical in the development of T-cell-mediated immunity, paramount for viral clearance. Thus, an increased production of serum IFN-γ has been strongly linked to self-limited HCV infection[11,12
]. Our data suggest an increased production of IFN-γ in the patients treated with Viusid when compared with baseline, maybe suggesting a reestablishment of the T-cell-mediated immunity via
Th1-type response; however, the underlying mechanisms by which Viusid is able to induce IFN-γ are still poorly understood. On the other hand, IL-10 is a Th2-type response cytokine with a suppressive effect on the generation of Th1-type response cytokines (IFN-γ and IL-12), which have recognized anti-inflammatory and slight proviral effects[13-15
]. Our data show that serum IL-10 levels were markedly increased with the administration of Viusid in spite of simultaneously increasing serum IFN-γ levels. IL-10 is known to suppress the secretion of pro-inflammatory cytokines, such as TNF-α, IFN-γ, and IL-12[15,39,40
]. In contrast, administration of neutralizing monoclonal antibodies to anti-IL-10 enhances in vitro
IFN-γ production in HCV patients[41
], suggesting that IL-10 levels differ widely between individuals, possibly due to polymorphisms in the promoter region of the IL-10
Recent data support the hypothesis that long-term IL-10 therapy to treat chronically HCV-infected patients leads to significant histological improvement of inflammation and fibrosis[15
Our data suggest that increased serum IL-10 levels could be a significant finding to explain the effect of Viusid on histological features (inflammation and fibrosis) in patients with CHC and non-alcoholic fatty liver disease (NAFLD)[27,31
Recent studies have indicated that glycyrrhizin, an aqueous extract of licorice root and the main ingredient of Viusid, can induce a significant production of IL-10 by liver dendritic cells in concanavalin A-induced hepatitis in mice[43
Our findings also show that a short course of Viusid slightly reduces IL-1α and stabilizes TNF-α concentrations in patients with CHC. These are markers of inflammation and fibrosis that are elevated in HCV-infected subjects compared with healthy controls[16,17,38
]; therefore, their reduction or stabilization could be associated with histological improvement, in particular inflammation and fibrosis.
Viusid has chemical ingredients with recognized immune regulatory properties that would explain in part the effect on cytokine secretion. Zinc supplementation has demonstrated that it can significantly increase IFN-γ and IL-10 secretion and reduce IL-1 and TNF-α secretion in patients with chronic inflammatory diseases[44
GGT has been regarded as a biomarker of hepatobiliary disease and alcohol consumption/abuse[45
]. In addition to liver disease, GGT has been associated with high all-cause mortality, cardiovascular disease incidence and death, and cancer incidence and death[46,47
]. Accumulating experimental evidence suggests that GGT is a key mediator in the mechanisms of oxidative stress. GGT-mediated oxidative stress has been reported capable of inducing lipid oxidation, oxidation of protein thiols, alterations of the normal protein phosphorylation patterns, and biological effects such as the activation of transcription factors[48
In the current study, we found that serum GGT levels were significantly reduced in the patients treated with Viusid in comparison with unchanged levels in the patients assigned to placebo. The mechanism that explains the contribution of Viusid to GGT reduction has not been fully elucidated and further studies are needed to evaluate the clinical impact of long-term Viusid administration in patients with other forms of chronic liver disease, cardiovascular disease, metabolic syndrome, and cancer.
On the other hand, the administration of Viusid did not improve aminotransferase levels during the 24 wk of treatment. In contrast to what has been suggested in two recently published papers[33,37
], Viusid without supportive therapy such as antiviral therapy or lifestyle modification for CHC and NAFLD, respectively, is unable to induce improvement in aminotransferase levels, regardless of its proven and significant antioxidant and immunomodulatory capability. Further studies must evaluate higher doses and long-term administration of Viusid to determine its effect on aminotransferase levels in patients with chronic liver diseases.
The main strength of this study is the presence of a concurrent placebo control group allowing adequate comparison between control and experimental groups for outcome measures. A possible weakness of our study is that patients were treated for 24 wk only, and it remains unclear whether some parameters related to oxidative stress and immunological status can be improved beyond 24 wk.
In conclusion, our data suggest that Viusid improves oxidative stress through reduction of lipid peroxidation products and that it has an immunomodulatory effect on cytokine secretion via increased production of IFN-γ and IL-10, decreased production of IL-1α, and stabilized TNF-α secretion in patients with CHC who have failed previous antiviral treatment. Our findings also highlight the limitations in our understanding of the complex mechanism of the host immune response and its interaction with pro-oxidant/antioxidant status.