**6. Laennec and Porcine also have the effects of anti-inflammation and immune modulation in the treatment of chronic liver diseases**

For the clinician, the serum elevation of ferritin in chronic liver diseases (CLD), with the exception of hemochromatosis, is always assumed to be a non-specific marker of hepatic inflammation and not of iron overload [88]. It is generally considered that iron is a putative component that interacts with oxygen radicals, and high rates of hyperferritinemia together with increased hepatic iron stores have been demonstrated in NASH [22].

In the progression and pathogenesis of NASH, the role of hepatic iron still remains debatable. It is empirically accepted that iron-restricted diets or phlebotomy reduce hepatic damage as well as insulin resistance (IR) in patients with NAFLD/NASH [89]. However, the exact mechanisms involved in iron accumulation in NASH remain unresolved. Several mechanisms such as dysregulation of iron-regulatory molecules, genetic factors linked to IR and erythrophagocytosis by Kupffer cells might be responsible for hepatic iron overload in NASH [90].

A simple clinical study of phlebotomy treatment was reported by Chakraberti and Adams, performed on a series of 56 patients with histology-proven NAFLD [91]. Liver biopsy and liver iron concentration (LIC) were evaluated at entry and 6 months after phlebotomy. They did not find any significant correlation between hepatic inflammation as measured by NAS score, LIC and the level of serum ferritin and other genetic markers of inflammation, such as ESR and CRP. The authors had the conclusion that elevated serum ferritin is associated with hepatic iron accumulation, but that liver inflammation is not the cause of increased serum ferritin in patients with NAFLD.

Recently, other authors [92] demonstrated that elevated ferritin levels reflect iron stores, and not hepatic inflammation, being predictors of vascular damage in NAFLD. Irrespective of the underlying mechanisms, the only certainty is that an increased serum ferritin level in NAFLD is not a marker or a cause of inflammation, but a consequence of iron accumulation within the hepatocyte.

The resident macrophages in the sinusoids of the liver, Kupffer cells, have been widely implicated in hepatic injury such as endotoxin–mediated liver injury. Kupffer cells are known to express the death ligands, tumour necrosis factor α (TNF α), TNFrelated apoptosis-inducing ligand (TRAIL), and Fas ligand [93, 94].

As a consequence of chronic tissue damage, HSCs, as well as other extracellular matrices – producing cells such as fibroblasts and myofibroblasts – undergo a process of activation towards a phenotype characterised by increased proliferation, motility, contractility and synthesis of extracellular matrix components. HSC activation is regulated by several soluble factors, including cytokines, chemokines, growth factors and products of oxidative stress as well as by extensive changes in composition and organisation of extracellular matrix components. Controlled cell death (apoptosis) could also be a mechanism underlying the termination of HSC proliferation. Spontaneous apoptosis is detected in parallel with HSC activation. Hepatic fibrosis is a complex dynamic process mediated by the death of hepatocytes and the activation of HSCs. Lipid peroxidation including the generation of ROS, TGF-β and TNF-α can be implicated as a cause of hepatic fibrosis.

The major source of ROS production in hepatocytes is NADH and NADPH oxidases localised in mitochondria. NADH and NADPH oxidases leak ROS as part of their operation. Hepatic fibrosis itself causes no symptoms but can lead to end-stage cirrhosis. In cirrhosis, the failure to properly replaced destroyed hepatocytes and the excessive collagen deposition to distort blood flow through the liver (portal hypertension) results in severe liver dysfunction.

Under physiological conditions, hydrogen peroxide plays an important role in intracellular signalling. In terms of pathological actions, ROS participate in the development of liver diseases. In this situation, hydrogen peroxide is converted into the hydroxyl radical, which is a harmful and highly reactive ROS, in the presence of transition metals such as iron. At the cellular levels, origin of hepatic fibrosis is initiated by the damage of hepatocytes, followed by the accumulation of neutrophils and macrophages including Kupffer cells on the sites of injury and inflammation in the liver. When hepatocytes are continuously damaged, leading to cell death, the

production of extracellular matrix proteins such as collagens predominates over hepatocellular regeneration. Overproduced collagens are deposited in injured areas instead of destroyed hepatocytes.

Judging from our clinical data presented before [73–75], Laennec and Porcine could have improved chr. liver diseases (CHC, NASH, PBC, AIH etc.) through the mechanisms of anti-inflammatory and immune modulative actions, which were evidenced through the fundamental examinations performed by Shindo et al. [62].
