**4. Ameliorating effects of 'hepcidin inducer Laennec and Porcine' for the progression of NASH/NAFLD**

NASH is a severe form of fatty liver disease that is defined by the presence of inflammation and fibrosis, ultimately leading to cirrhosis and hepatocellular carcinoma. Shindo et al. [62] evaluated the effect of human placenta extract (HPE) and Laennec treatment in a mouse model of NASH. In the methionine- and choline-deficient (MCD) diet-induced liver injury model, fibrosis started in the regions around the sinusoids.

They dispensed the MCD diet with high-salt loading (add 8% NaCl in the drinking water) to mice deficient in the vasoprotective molecule RAMP2 for 5 weeks, with or without HPE. In both the HPE and control groups, fibrosis was observed in regions adjacent to the sinusoids, but fibrosis was not so pronounced in the HPE-treated mice. Levels of TNF-α and MMP9 expression were also significantly reduced in

*Regulation of Iron Metabolism in NAFLD/NASH DOI: http://dx.doi.org/10.5772/intechopen.107221*

HPE-treated mice, and oxidative stress was suppressed in the perivascular region. These observations indicate that HPE ameliorates NASH-associated pathologies by suppressing inflammation, oxidative stress and liver fibrosis.

HPE has been prescribed clinically to treat chronic hepatitis, liver cirrhosis and other hepatic diseases for more than 40 years in Japan. In an experimental animal model of hepatitis, HPE reportedly ameliorated hepatic injury through liver regeneration and inhibition of inflammatory reactions and hepatocyte apoptosis [70, 71]. Moreover, Shimokobe et al. recently reported that HPE is effective in NASH patients who were unresponsive to lifestyle intervention [72].

As for histopathological changes in the liver, silver staining histological sample revealed fibrotic areas adjacent to the sinusoids in both groups; however, the fibrosis was not so severe in HPE-treated mice. By using immunofluorescent staining, the authors observed high expression levels of p67 phox, a cytosolic component of NADPH oxidase, in the perivascular regions of all mice; however, the expression levels were less marked in HPE-treated mice. Levels of 4HNE, a lipid peroxidation product, were also decreased in HPE-treated mice. Judging from these observations, it is indicated that HPE treatment for ameliorated liver injury is possible by reducing inflammation, oxidative stress and fibrosis.

In an earlier study, HPE was shown to suppress inflammation in a chronic arthritis rat model using complete Freund's adjuvant [73]. Direct effects of HPE on the production of pro-inflammatory cytokines and mediators have also been reported. For example, HPE reportedly inhibits the production of nitric oxide, TNF-α and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7 macrophages [74]. In the present study, the authors found that HPE significantly suppressed TNF-α expression in an MCD fed-diet model. This suggests that HPE may suppress the progression of chronic inflammation initiated by lipid accumulation within hepatocytes of the NASH patients.

#### **Figure 1.**

*The most important basic data is expressed. Laennec induces the expression of hepcidin mRNA both in rat primary hepatocyte and HepG2 cells in a dose dependent manner; this means that in the human body, the possibility of hepcidin mediated action of Laennec might be the main route or at least one of the important mechanism of its efficacy in regulating iron metabolism.*

Besides inflammation, oxidative stress seems to also contribute to chronic liver injury. In that regard, HPE showed both anti-oxidative and anti-inflammatory activities in rats exposed to benzopyrene (BaP) [75]. Application of H2O2 to cultured cells is performed to evaluate the cellular damage caused by oxidative stress. It is also observed that serum hepcidin levels are typically elevated in individuals with NASH [76]. As this in itself fails to explain iron loading in NASH, one might consider that dysregulated iron metabolism occurs in NASH independently of hepcidin.

The contribution of adipose tissue-derived hepcidin to the serum hepcidin pool is uncertain, however, this is another potential factor that may explain the increased serum hepcidin levels in NASH. Further complexity in these relationships arises when one considers that iron deficiency has been shown to be associated with obesity, and in women with obesity and NAFLD [77, 78]. Together, these findings suggest that the interaction between iron and lipid metabolism is multi-faceted. It seems that 'just enough' but 'not too much' iron may be critical for preventing dysfunctional lipid metabolism.

Previous studies [73–75] have revealed that the administration of Laennec significantly improved T2DM complicated with NASH and other chronic liver diseases, suggesting the importance of iron regulation on insulin-resistant T2DM showing hyperferritinemia.

Thus, more experimental and clinical studies are required to confirm or refute the claim that hepcidin has a role in T2DM. To shed light on the factors that alter hepcidin expression, the authors performed experiments with HepG2 and HuH7, human hepatoma cell lines that are widely used for this purpose. Despite the considerable advances made recently, further explorations are required to investigate the cellular mechanisms and functions of peripheral hepcidin, as well as its regulation in different organs (**Figures 1** and **2**).

### **Figure 2.**

*Systemic iron homeostasis is regulated by hepcidin. Judging from the clinical data of Laennec, formerly obtained through the treatment of chronic liver diseases such as NASH and chronic hepatitis type C, the sites of action of Laennec resemble those of hepcidin. Hepcidin is also expressed in the pancreas of rats and humans, which means that pancreas is an iron-regulating organ beyond their proper glucose–regulatory function. According to basic data, the regulation of hepcidin expression is similar in the liver and in the endocrine pancreas.*
