bubble_chart Overview

Liver fibrosis refers to the excessive deposition of fibrous connective tissue in the liver, which is the result of an imbalance between fibrogenesis and fibrolysis. Fibrogenesis is a repair response of the body to injury. Repeated or persistent chronic inflammation and necrosis of the liver parenchyma caused by various diseases can lead to continuous fibrogenesis in the liver, resulting in liver fibrosis. From the clinical and pathological evolution of many chronic liver diseases, especially chronic viral hepatitis, liver fibrosis and cirrhosis are a continuous developmental process, and the two are difficult to separate distinctly.

bubble_chart Etiology

The mechanism of hepatic fibrosis

Hepatic stellate cells are the primary source of various extracellular matrices. During the initiation and progression of hepatic fibrosis, stellate cells are activated and transformed into myofibroblast-like cells and fibroblasts, making the activation process of stellate cells a focal point in the study of hepatic fibrosis mechanisms. Factors regulating stellate cells can be divided into insoluble and soluble categories, the former being various extracellular matrices, and the latter including various growth factors and cytokines.

The functional basement membrane (type IV collagen and laminin) beneath the sinusoidal endothelium in a normal liver plays a crucial role in maintaining the quiescent state of stellate cells (storing Vit. A, secreting type IV collagen). Once the basement membrane is damaged, the phenotype of stellate cells can change.

There are numerous cytokines and growth factors that regulate stellate cells. Those that promote their proliferation include PDGF, EGF, IGF-1, FGF-2, TGF-α; those that inhibit their proliferation include retinol, retinoic acid, and endothelin; TGF-β itself inhibits the growth of stellate cells, but by stimulating the expression of PDGF and FGF-2, it can also promote their growth. Among these cytokines, TGF-β ₁

and PDGF have been studied more extensively. In the liver, TGF-β ₁ is mainly produced by stellate cells, Kupffer cells, endothelial cells, platelets, and hepatocytes, exerting regulatory effects on other cells through paracrine signaling. TGF-β ₁ promotes the expression and secretion of type I, III, IV collagen, fibronectin, tenascin, chondronectin, thrombospondin, biglycan, and decorin by stellate cells. It also promotes its own expression through autocrine action, inhibits the expression of metalloproteinases (collagenase, stromelysin) and plasminogen activators, and promotes the expression of plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitors of metalloproteinases (TIMP) by endothelial cells and stellate cells. Therefore, TGF-β ₁ is currently recognized as the most important profibrogenic cytokine. PDGF can be produced by various cells. It is a potent mitogen that promotes the proliferation of stellate cells through α and β receptors, and TGF-β ₁ can enhance this effect; it itself has no significant effect on the expression of extracellular matrix. TNF-γ is produced by lymphocytes and inhibits the activation, growth, and collagen secretion of stellate cells. In the liver, ET-1 is mainly produced by sinusoidal endothelial cells and activated stellate cells. Stellate cells express high levels of ET-1 receptors, and through paracrine and autocrine actions, they cause the contraction of activated stellate cells and collagen fiber bundles, leading to changes in sinusoidal blood flow and distortion of the hepatic lobule structure. The three-stage hypothesis of stellate cell activation.

I. Preinflammatory Phase

When the liver parenchyma is injured, the permeability of the cell membrane increases, releasing "injury hormones" (Wound Hormone), which activate fat-storing cells through paracrine action. In vitro experiments have shown that rat liver cells can release cytokines that promote the proliferation of fat-storing cells—Hepatocellular Ito Cell Initiator (Hepitoin). Additionally, if the injury to the liver stroma damages the functional basement membrane beneath the sinusoidal endothelium, it can also promote phenotypic changes in fat-storing cells, leading to their activation.

II. Inflammatory Phase

Due to liver parenchymal injury, Kupffer cells, mononuclear macrophages, lymphocytes, and platelets are activated, releasing various inflammatory mediators and cytokines, such as acute-phase proteins (IL-1, IL-6, TNF-α) and TGF-1, EGF, PDGF, TGF-α, TGF-β, etc. These factors also promote the proliferation of stellate cells and fibrogenesis through paracrine pathways. During this phase, stellate cells are induced by certain cytokines secreted by Kupffer cells to express PDGF receptors, responding to PDGF by proliferating and activating into myofibroblast-like cells. TGF-β not only enhances the proliferative effect of PDGF on stellate cells but also promotes their expression and secretion of various extracellular matrices while inhibiting their degradation.

III. Postinflammatory Phase

In the aforementioned two phases, stellate cells activated through paracrine pathways express large amounts of TGF-α, TGF-β, FGF, and their receptors, continuously stimulating their own division and proliferation through autocrine actions and synthesizing and secreting various extracellular matrices such as collagen. They can also activate other stellate cells that are still in a "quiescent" state through paracrine actions. This mechanism explains why the process of liver fibrosis can continue to progress even after the primary stimulating factors are removed.

The role of matrix-degrading enzymes in the various stages mentioned above can be summarized as follows: type IV collagenase secreted by stellate cells disrupts the normal functional basement membrane of the sinusoidal endothelium, promoting its proliferation and activation; TGF-β inhibits the activity of interstitial collagenase and stimulates the activity of TIMP, thereby promoting the deposition of extracellular matrix.

bubble_chart Pathological Changes

Liver fibrosis refers to the excessive deposition of fibrous connective tissue in the liver, resulting from an imbalance between fibrogenesis (i.e., increased synthesis of extracellular matrix) and fibrolysis (i.e., degradation of extracellular matrix). Fibrogenesis is a repair response of the body to injury, and once the harmful factors are removed and the extracellular matrix components are restored, fibrogenesis ceases. Therefore, even severe acute or transient liver diseases do not lead to liver fibrosis. However, repeated or persistent chronic liver parenchymal inflammation and necrosis caused by various disease factors can lead to continuous fibrogenesis in the liver, resulting in liver fibrosis. Further progression can lead to the destruction of liver lobule structure and nodule formation, which is known as liver cirrhosis. From a pathological perspective, the presence of diffuse liver fibrosis without nodule formation (e.g., congenital liver fibrosis, fibrosis in the third zone of the liver acinus due to heart failure) cannot be termed liver cirrhosis; similarly, the presence of nodule formation without diffuse liver fibrosis (e.g., focal nodular hepatocellular hyperplasia, nodular regenerative hyperplasia of hepatocytes) is also not liver cirrhosis. However, from the clinical and pathological evolution of many chronic liver diseases, especially chronic sexually transmitted viral hepatitis, liver fibrosis and cirrhosis are a continuous developmental process, and the two are difficult to separate completely. In recent years, with the rapid development of cell biology and molecular biology, our understanding of the composition, metabolism, and biological functions of the liver's extracellular matrix has deepened, leading to significant progress in the research on the mechanisms, diagnosis, and treatment of liver fibrosis.

I. Composition, Metabolism, and Biological Functions of the Extracellular Matrix (ECM)

The extracellular matrix generally includes collagen, non-collagen glycoproteins, proteoglycans, and elastin. According to the latest perspective, ECM should also include interstitial metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), matrix adhesion molecules (i.e., extracellular matrix receptors), and various growth factors and cytokines bound to the macromolecules of interstitial components. It is now believed that ECM is not a disorganized, static substance that merely serves as a scaffold, but rather a well-organized, metabolically active life molecule that significantly influences the structure and function of cells, tissues, and organs in terms of morphology, growth, differentiation, and metabolism.

(1) Collagen

Collagen is the most important component of the extracellular matrix, and at least 19 types of collagen with different gene sequences have been identified so far. In the liver, the five types with higher total content are: Type I (33%), Type III (33%), Type IV (1%), Type V (1-10%), and Type VI (0.1-1%). The collagen content in a normal human liver is approximately 5.5 mg/g of liver wet weight, with a Type I/Type III collagen ratio of 1:1. During liver fibrosis and cirrhosis, the collagen content in the liver can increase severalfold, and the Type I/Type III ratio also increases, reaching up to 2.38 in the late stage [third stage]. Based on the morphology, structural characteristics, and distribution of collagen, it can be divided into two major categories: ① Fibrous collagen: including Types I, III, V, and VI. Type V collagen, after the removal of the C-terminal peptide, is distributed around the sinusoids and portal areas, serving as a core that allows Types I and III collagen to form thick fibers. Type VI collagen, with a bead-like structure, is distributed between the fiber bundles formed by Types I, III, and V collagen, playing an adhesive role. ② Basement membrane collagen: specifically Type IV collagen, whose terminal peptides are not removed but instead connect to form a three-dimensional mesh-like structure, primarily distributed beneath the liver sinusoid endothelium, serving as the main component of the functional basement membrane for hepatocytes and endothelial cells. The synthesis of collagen involves steps such as gene transcription, translation, post-translational modifications (hydroxylation and glycosylation) to form α-peptide chains. Three α-chains form a triple helix, which is the basic unit of collagen—precollagen. This is then secreted into the extracellular space via microtubules, where the N-terminal and C-terminal peptides are cleaved, allowing the collagen to cross-link and form collagen fibers or mesh-like structures.

(2) Noncollagenous Glycoproteins

are another important component of the extracellular matrix. Multiple functional domains within their molecules can bind to other extracellular matrix components and various transmembrane protein receptors on cell membranes, thereby influencing cell growth, differentiation, metabolism, and other biological behaviors. There are many types, and the main ones are listed below:

1. Fibronectin

can be divided into plasma (soluble) and cellular (insoluble) types. The former is mainly produced by hepatocytes, while the latter is primarily produced by fat-storing cells, macrophages, and vascular endothelial cells. Its content increases in the early stages of liver fibrosis, serving as a scaffold for subsequent collagen deposition.

2. Laminin

is a cross-shaped structure composed of three subunits (one α chain and two β chains). Its molecule contains functional domains that can bind to cell surface receptors and heparin. Together with type IV collagen, it forms the main component of the basement membrane, distributed on the basement membranes of blood vessels and bile ducts, with a small amount also found in the subendothelium of liver blood sinuses. This is significant for maintaining the differentiated state of cells.

3. Tenascins (Cytotactin, Bronionectin, Hexabronchions)

is a hexamer composed of six identical subunits, produced by fat-storing cells. In liver fibrosis, it deposits in areas with severe cell injury, such as around the central vein and within fine fibrous septa, and may play a role in the early deposition of ECM.

4. Undulin

is composed of three subunits, with its monomer resembling laminin but lacking one short arm. It is mainly distributed among tightly packed type I and III collagen fibers and plays an important role in maintaining the supramolecular structure of collagen. Its cellular origin is still unclear.

5. Thrombospondin

is formed by the ends of three identical subunits creating a small sphere in the center, capable of binding heparin, type V collagen, fibronectin, plasma plasminogen, and fibrinogen. Its function is to prevent cell spreading.

6. Vitronectin (VN)

is produced by hepatocytes and has a distribution similar to fibronectin, with the ability to bind collagen. In liver cirrhosis, it is distributed in areas of collagen fiber deposition. Plasma VN levels show some correlation with the degree of liver fibrosis.

7. Entactin (Nidogen)

is asymmetrically dumbbell-shaped and can specifically bind to the central region of laminin via covalent bonds, thereby regulating the binding of laminin to its receptors. Its distribution is similar to that of laminin units, mainly found in the basement membrane.

(3) Proteoglycan

Proteoglycans are a class of molecules with a protein core skeleton, to which glycosaminoglycans are attached at N or O positions.

Glycosaminoglycan (GAG) side chains are macromolecular substances that, along with collagen, are distributed in the extracellular matrix and basement membrane, as well as on the cell membrane. The glycosaminoglycans can be divided into Heparan Sulfate, Dermatan Sulfate, Chondroitin Sulfate, and Keratan Sulfate. In a normal liver, Heparan Sulfate is the most abundant, accounting for 60% of the total GAG in the liver; during liver fibrosis, its content decreases, while the contents of Dermatan Sulfate and Chondroitin Sulfate increase. Hyaluronic Acid is a unique polysaccharide that is not sulfated and lacks a protein backbone. In recent years, the cDNA of many proteoglycan core proteins has been cloned, leading to a deeper understanding of their biological functions. Based on their distribution, proteoglycans can generally be divided into two categories:

1. Proteoglycans associated with the cell membrane

Their core proteins often have transmembrane functional domains, and some can also be connected to the cell membrane through their GAG side chains. They contain a large amount of heparan sulfate and a small amount of chondroitin sulfate, and can bind to extracellular matrix, growth factors, cell adhesion molecules (CAM), and protease inhibitors, enhancing or weakening the activity of these substances.

Syndecan is produced in liver tissue and cultured hepatocytes, and serves as a receptor for some interstitial cells. It can bind to type I, III, and V collagen, fibronectin, and common bletilla tuber cell adhesion molecules through its GAG side chains. It also acts as a coreceptor for bFGF. Its main function is signal transduction.

Thrombomodulin is produced by vascular endothelial cells, and its GAG side chain is chondroitin sulfate. It can bind to thrombin, inhibit the activation of fibrinogen and factor V, thereby regulating the blood coagulation process.

Betaglycan has GAG side chains of chondroitin sulfate and heparan sulfate. It is a cell membrane receptor with high affinity for TGF-β and serves as its type III receptor.

2. Proteoglycans associated with the extracellular matrix

They are mainly distributed in the extracellular matrix.

Fibromodulin has a GAG side chain of sulfur tamarind pulp, which can regulate the formation of collagen microfibrils. It has not been found in the liver.

Perlecan is produced by non-parenchymal cells and is distributed in the basement membrane and bile ducts, blood vessels. It can bind to endothelial cells and hepatocytes.

Decorin has GAG side chains of chondroitin sulfate and dermatan sulfate. It can bind to type I and IV collagen and fibronectin, delaying the formation of collagen microfibrils. TGF-β can increase the expression of Decorin, while Decorin can inactivate TGF-β. In vitamin A-rich stellate cells, Decorin expression is higher, thus inhibiting TGF-β activity; in vitamin A-deficient stellate cells, Decorin expression is reduced, thus weakening the inhibition of TGF-β.

Biglycan has GAG side chains of chondroitin sulfate and dermatan sulfate, produced by stellate cells and myofibroblasts. It binds to TGF-β through its core protein and to bFGF through its GAG. TGF-β can increase its expression, while retinoic acid can decrease its expression.

Versican has side chains of chondroitin sulfate, and its expression can be increased by PDGF and TGF-β. It can bind to hyaluronic acid. It has not been determined whether liver cells can produce Versican.

(4) Cell-Matrix Adhesion Molecules

Molecular interactions between cells and extracellular matrix components are mediated by specific membrane proteins, namely various adhesion molecules. Some adhesion molecules frequently expressed on other epithelial and endothelial cells are not expressed in normal hepatocytes and sinusoidal endothelial cells, but their expression is upregulated during the process of fibrosis. Cell-matrix adhesion molecules mainly include the Integrin family and the CD₄₄ protein family.

1. Integrin

is a dimer composed of non-covalent bonds between α and β chains. Currently, 17 types of α chains and 8 types of β chains are known to form 21 functional dimers. As receptors, Integrins can bind to multiple ligands, and a single extracellular matrix component can be bound by various Integrins through different recognition sites. The most important recognition site is the R-G-D (Arg-Gly-Asp) sequence in extracellular matrix molecules, which can be recognized by most Integrin receptors. Each Integrin has its specific tissue distribution, and a single cell can express multiple Integrins, with overlapping ligand recognition sites. In non-polarized cells, Integrins are distributed on all surfaces, while in polarized cells, they are only found on the basal and lateral surfaces. They not only function in adhesion but also, due to their intracellular domains being connected to the cytoskeleton, can transmit signals that promote cell proliferation, differentiation, and migration. Based on the β chain, Integrins can be divided into 3 subfamilies:

β₁ (10 types), β₃ (2 types), β₄ (1 type) Their main function is to mediate cell-matrix adhesion and are distributed in most somatic cells. They can bind to collagen and non-collagen glycoproteins, but no binding to proteoglycans has been found.

β₂ (3 types), β₇ (2 types) are mainly expressed in leukocytes to mediate cell-cell interactions within the immune system.

β₆ (1 type), β₇ (1 type), β₈ (1 type) have limited distribution and their functions are not yet clear.

2. CD₄₄ protein family

is a class of heavily glycosylated transmembrane proteins widely distributed in somatic cells, mediating cell-cell and cell-matrix interactions. There are many members in this family, and under normal conditions, CD₄₄ protein expression is not detected in hepatocytes but can be expressed in sinusoidal endothelial cells.

The most significant change in adhesion molecules during liver fibrosis is the induced expression of laminin receptors in the Integrin family in hepatocytes and sinusoidal endothelial cells, and this upregulation occurs before the formation of cirrhosis. Profibrotic cytokines such as TGF-β and pro-inflammatory cytokines such as TGF-α, IL-1β, IFN-γ can promote the high expression of Integrin receptors and CD₄₄ proteins. Regardless of the underlying disease, the induced expression of Integrin receptors is similar, suggesting a non-specific response to changes in the extracellular matrix components adjacent to cells. Activation of Integrin receptors can directly and indirectly trigger a series of intracellular events, such as cell morphology, generation and transformation, metabolism, secretion, and tyrosine phosphorylation of cytoplasmic substrates, indicating that the extracellular matrix plays an important role in regulating the structure and function of hepatocytes and sinusoidal endothelial cells. In the early stages of liver fibrosis, the expression of Integrin receptors may promote the local deposition and organization of the extracellular matrix, so the upregulation of these cell-matrix adhesion molecules is not only an adaptive response to changes in the cellular microenvironment but also likely plays an active role in initiating the fibrosis process itself.

(5) Matrix Metalloproteinases (MMPs)

The extracellular matrix is mainly degraded by MMPs, and so far, 9 types of MMPs have been discovered, which can be divided into three major groups based on their substrates: collagenases, gelatinases, and stromelysins. Their substrate specificity and cellular sources are shown in the table.

Table: Substrate specificity and cellular sources of matrix metalloproteinases

These matrix metalloproteinases belong to a gene family, and their structures contain several highly conserved common functional regions in amino acid sequences, such as the propeptide functional region (containing the PRCGVPDV sequence) that maintains the latency of the zymogen, the 120-amino acid N-terminal functional region, and the zinc-binding functional region (containing the HELGH sequence) at the catalytic site. The regulation of matrix metalloproteinases can occur at three levels: gene transcription regulation, activation of inactive zymogens, and inhibition of activated enzymes by tissue inhibitors.

Some growth factors, cytokines, and retinoic acids collectively regulate the expression of multiple matrix metalloproteinases, while others specifically regulate the expression of a particular matrix metalloproteinase.

Matrix metalloproteinases are secreted in an inactive zymogen form and must be hydrolytically activated in the extracellular space to degrade the extracellular matrix. Urokinase-plasminogen activator (UPA) or tissue-plasminogen activator (TPA) converts plasminogen into plasmin, which then activates stromelysin and partially activates procollagenase. The activated stromelysin further activates collagenase. This cascade of activation is regulated by plasminogen activator inhibitor-1 (PAI-1). Cells that synthesize matrix metalloproteinases often also synthesize plasminogen activator inhibitor to prevent excessive degradation of the matrix.

(VI) Tissue Inhibitor of Metalloproteinases (TIMP)

The activated matrix metalloproteinases are regulated by specific tissue inhibitors of metalloproteinases in the extracellular space. Three types of TIMPs have been discovered, each of which can directly bind to the catalytic site and inhibit the activity of all matrix metalloproteinases. However, TIMP-1 primarily inhibits interstitial collagenase, stromelysin, and gelatinase B (92KD type IV collagenase), while TIMP-2 mainly inhibits gelatinase A (72KD type IV collagenase). Recently, it has been found that TIMP can also bind to the carboxyl terminus of certain pro-metalloproteinases, preventing their activation.

(VII) Cytokines and Growth Factors

The extracellular matrix can serve as a storage site for cytokines and growth factors. When other extracellular matrix components undergo changes, they regulate the activity of cytokines through various extracellular matrix adhesion molecules. Cytokines, in turn, can promote the synthesis or degradation of other extracellular matrices.

II. Cellular Sources of Extracellular Matrix

Liver cells can be divided into: ① parenchymal cells, i.e., hepatocytes; ② non-parenchymal cells, including fat-storing cells (Ito cells), sinusoidal endothelial cells, Kupffer cells, and pit cells. Through in vitro cell culture, immunohistochemistry, and molecular in situ hybridization studies, a better understanding of the sources of extracellular matrix has been achieved. It was previously thought that collagen was mainly synthesized by hepatocytes, but it was later found to be primarily produced by fat-storing cells; moreover, other extracellular matrices are also mainly produced by fat-storing cells. The cellular sources of extracellular matrix are summarized in the table.

Table: Cellular Sources of Extracellular Matrix

bubble_chart Auxiliary Examination

1. Clinical and Imaging Diagnosis

Any chronic liver disease, especially chronic sexually transmitted disease viral hepatitis patients, have the potential to develop liver fibrosis and cirrhosis. Therefore, it is essential to carefully inquire about the medical history and conduct a comprehensive physical examination for each such patient to detect early signs of cirrhosis. Modern imaging techniques such as B-mode ultrasound, CT, and magnetic resonance imaging (MRI) can reveal thickening of the liver membrane, irregular or nodular liver surface contours, uneven enhancement of liver parenchyma echoes or increased CT values, changes in lobe proportions, increased spleen thickness, and widening of the portal vein and splenic vein diameters. Color Doppler ultrasound or radionuclide scanning can measure liver stirred pulse and portal vein blood flow as well as functional portosystemic shunting.

2. Histopathological Examination

To date, histopathological examination through liver puncture or laparoscopic liver biopsy remains the "gold standard" for diagnosing liver fibrosis and cirrhosis. In 1994, the new international grading and staging criteria for chronic hepatitis suggested using liver fibrosis as the basis for disease staging, with separate scores for grading (mainly the degree of inflammation and necrosis). Corresponding grading and staging recommendations have also been proposed domestically. Routine HE staining and various extracellular matrix histochemical, immunohistochemical, and even molecular in situ hybridization techniques allow us to obtain more information about liver fibrosis from liver tissue samples; computer image analysis and other technologies can provide quantitative data to observe the effects of anti-fibrotic treatments. However, liver biopsy techniques also have their limitations, as although chronic hepatitis and other lesions are diffuse, it is difficult to ensure that a single sample reflects the entire liver, especially in cases of liver fibrosis and cirrhosis where fine-needle aspiration biopsy may not yield sufficient samples, posing certain diagnostic difficulties. For drug evaluation, dynamic serial liver biopsy histopathological examinations should be conducted.

3. Serological Diagnosis of Liver Fibrosis

Given the limitations of liver biopsy histopathological examination, efforts have been made to find serological markers to monitor the progression of liver fibrosis and evaluate the efficacy of anti-fibrotic treatments. Ideal serological markers for liver fibrosis should meet several criteria: ① high liver specificity; ② ability to assess the process of liver fibrosis or degradation, reflecting the degree of liver fibrosis and the disorder of liver parenchymal and stromal structures; ③ not taken up by sinusoidal endothelial cells, and not excreted through the biliary tract or kidneys; ④ simple and easy measurement methods with high sensitivity. In reality, existing tests for measuring extracellular matrix components in serum do not fully meet these criteria, but animal experiments and clinical pathological studies have identified several valuable markers for assessing liver fibrosis. Overall, these markers show good correlation with corresponding extracellular matrix components and their mRNA levels in the liver in animal experiments; in clinical studies, these markers also show some association with the degree of liver fibrosis in histopathology, being higher in chronic active liver disease (so-called chronic active hepatitis and active cirrhosis) than in inactive liver disease (chronic persistent hepatitis and inactive cirrhosis). However, there is considerable overlap between groups, making it difficult to make a definitive diagnosis based on a single result; the combined use of multiple markers and dynamic measurements may be more helpful in assessing the relative strength of fibrosis and fibrolysis. Below are a few serological markers for liver fibrosis that have been extensively studied.

1. Serum Type I Collagen (CI) and Type I Procollagen Carboxy-Terminal Peptide (PICR)

The liver contains a high amount of type I collagen, which significantly increases during the advanced stages of liver fibrosis and cirrhosis. The serum levels of this collagen show a high correlation with the degree of liver fibrosis. A RIA method has been established to determine the upper limit of serum CI in normal individuals, which is 197μg/L (M±2SD). It does not increase in acute hepatitis but rises in various chronic active liver diseases, with a correlation coefficient of 0.67 (P < 0.0001) with liver fibrosis resolving accumulation, but shows no correlation with liver inflammation indices. In chronic hepatitis and alcoholic liver disease, if serum CI is above 300μg/L, the specificity for diagnosing cirrhosis is very high. In patients with alcoholic liver disease, serum CI levels increase and serum PICP levels decrease after cessation of alcohol consumption, while serum CI levels do not change significantly, suggesting that PICP can reflect liver fibrogenesis, whereas CI reflects the deposition of liver protofibrils. However, both CI and CPCP are significantly influenced by bone metabolism, resulting in poor liver specificity, which limits their clinical application.

2. Serum Type III Collagen (CIII) and N-terminal Propeptide of Type III Procollagen (PIIINP)

Some foreign studies have used RIA to determine the serum CIII levels in normal individuals, which are up to 40μg/L (M±2SD). In chronic active liver disease, these levels are elevated, possibly due to the excessive secretion of new type III collagen from the gaps of sinusoidal endothelial cells into the bloodstream. The serum CIII levels correlate with liver tissue fibrosis (r=0.35, P<0.01) and also show some correlation with inflammatory activity. Domestic reports indicate that serum CIII (PCIII) levels are closely related to the degree of liver tissue fibrosis (r=0.984, P<0.01), and it has been proven that there is a correlation between serum PCIII and PIIIP in patients with chronic hepatitis and cirrhosis (r=0.510).

This is the most studied serological marker of liver fibrosis. It is the N-terminal peptide cleaved by peptidase after type III procollagen is secreted extracellularly. The collagen molecule forms only after the terminal peptides (N, C) are cleaved, and they cross-link to form microfibrils. Therefore, an increase in PIIINP reflects liver fibrogenesis. Animal experiments have shown that elevated serum PIIINP levels are closely related to increased collagen mRNA and TGF-β mRNA levels in liver tissue; clinical studies have also found a good correlation between serum PIIINP levels and the degree of liver tissue fibrosis, making it an indicator of liver fibrogenesis. Some also believe it is more closely related to the liver inflammation activity index, hence it also increases in acute hepatitis; additionally, its excretion requires uptake by liver sinusoidal endothelial cells, so serum PIIINP also increases in liver failure. Clinically, the test results should be interpreted according to specific conditions. We used a PIIIP RIA kit produced by a German company (now produced by a Japanese company) to measure serum PIIINP levels in 67 patients with various chronic liver diseases accompanied by grade I to IV liver fibrosis, which were 12.3±3.3ng/ml, 17.9±5.5ng/ml, 21.5±11.8ng/ml, and 32.9±12.7ng/ml, respectively, indicating a positive correlation between PIIIP and the degree of liver fibrosis. Notably, serum PIIIP levels in patients with either primary liver cancer or metastatic liver cancer are significantly higher than in other chronic liver disease patients, so for chronic hepatitis patients, if serum PIIIP remains abnormally high, the possibility of liver cancer should be alerted.

3. Serum Type IV Collagen (CIV) and C-terminal Peptide of Type IV Collagen (CIVCP, NCI) and N-terminal Peptide of Type IV Collagen (CIV NP, 7S)

Type IV collagen is deposited in the extracellular matrix without the need to remove terminal peptides during its anabolic process, so an increase in serum type IV collagen levels may reflect an accelerated turnover rate of the liver sinusoidal basement membrane. Both basic and clinical studies have found that serum type IV collagen levels are closely related to the degree of liver fibrosis and portal hypertension, with less relation to liver inflammatory activity.

Serum CIVCP (NC1) and CIVNP (7S) are also related to the degree of liver fibrosis. Since type IV collagen is deposited in the liver sinusoids, proliferating bile ducts, and the basement membrane around the limiting plate, the increase in serum CIVCP (NC1) and CIVNP (7S) reflects the degradation during the continuous remodeling of the basement membrane. These indicators can still increase even when fibrotic collagen (types I and III collagen) proliferation is not active in the advanced stage of liver fibrosis.

4. Serum Type IV Collagen (CIV)

Type IV collagen is distributed between large collagen fibers, and methods for measuring CIV such as RIA and ELISA have been developed. Molecular chromatography experiments have shown that the antigen detected in serum is a degradation product of CIV, making it an indicator of interstitial collagen degradation. When used in conjunction with PIIIP, an indicator of interstitial collagen synthesis, it provides a better understanding of the balance between fibrosis and fibrolysis. Another characteristic of this indicator is that it is not affected by human growth, making it also applicable to pediatric patients. However, serum CIV levels also increase significantly in cases of renal fibrosis and systemic connective tissue diseases, so clinical differentiation is necessary.

5. Serum Laminin P1 (Lam)

Lam is also a major component of the basement membrane. Serum Lam levels are associated with the degree of liver fibrosis and portal-hepatic venous pressure. The Laminin P1 RIA kit (currently produced by a Japanese company) was used to measure serum Lam in 71 patients with acute and chronic hepatitis. It was found that the levels in chronic active hepatitis and cirrhosis groups (3.42±1.13μ/ml, 6.58±1.05μ/ml) were significantly higher than in the chronic persistent hepatitis group (1.23±0.2μ/ml). Among 18 patients with chronic liver disease, those with portal hypertension (8.07±3.95μ/ml) had higher levels than those without (3.63±1.34μ/ml). It was also found that serum LamP1 was significantly elevated in acute hepatitis and primary liver cancer. These results are largely consistent with reports from foreign scholars. Some believe that the antigen used in commercial kits contains at least seven components, while the liver contains only one or two of these, so the increase in serum LamP1 levels is relatively small and not sensitive enough for diagnosing liver fibrosis. A new assay method using monoclonal double-antibody sandwich has been developed, which has higher specificity and sensitivity.

6. Serum Hyaluronic Acid (HA)

The increase in serum HA levels during liver fibrosis is partly due to increased synthesis by stellate cells, and partly due to liver sinusoid capillarization (endothelial cells losing HA receptors) and injury to liver sinusoid endothelial cells (losing the ability to metabolize HA), leading to reduced uptake and degradation of HA in the serum. Foreign scholars have found that serum HA levels in patients with chronic hepatitis C are positively correlated with liver fibrosis grading (r=0.47, P<0.01) and are not affected by age, with little relation to liver inflammation activity index. Using 90μg/L as the cut-off value, the specificity for diagnosing grade 2-3 liver fibrosis was 92%, with a sensitivity of 55%. Serum HA levels increase or decrease correspondingly with the progression or improvement of liver fibrosis. Domestically, Zhang Luyong et al. first established the RIA method for HA measurement, finding that HA levels were significantly elevated in patients with chronic active liver disease among 254 liver disease patients. Using 350ng/ml as the cut-off value, the sensitivity for diagnosing cirrhosis was 87.5%. In advanced stage cirrhosis, due to further impairment of liver sinusoid endothelial cell function, serum HA levels may be even higher, which differs from indicators like serum PⅢP that reflect active liver fibrosis.

7. Serum Fibronectin Receptor (Fn-R)

Fn-R is a member of the Integrin family and is overexpressed in the perisinusoidal cell membrane and fibrotic areas during liver fibrosis. Japanese scholars observed that serum Fn-R levels were significantly elevated in chronic hepatitis, cirrhosis, alcoholic liver disease, and hepatocellular carcinoma, correlating with histological fibrosis degree, suggesting it is a good serological indicator of liver fibrosis.

8. Undulin

Undulin is present on the surface of dense collagen fibers. When collagen structures are disrupted, it is released into the blood, making it, like type IV collagen, an indicator of collagen degradation.

9. Tenascin

Tenascin is mainly distributed in the perisinusoidal space of the liver. In early liver fibrosis, it deposits around central veins, within fine fibrous septa, and at the interface between extracellular matrix and hepatocytes. It may be an indicator of active liver fibrosis.

10. Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)

There are already ELISA kits available for measuring serum TIMP-1. Some have measured serum TIMP-1 in patients with chronic alcoholic liver disease and found that its levels are highly correlated with the degree of liver fibrosis. Moreover, there is less overlap among different groups of chronic liver diseases, making it an indicator reflecting low activity of extracellular matrix degradation in the liver.

11. Other Indicators

Since the metabolism of the extracellular matrix, particularly collagen, involves many enzymes, theoretically, it is possible to assess the balance between liver fibrosis and fibrolysis by measuring the enzymes that synthesize and degrade collagen. However, the methods for determining enzyme activity are cumbersome, influenced by many factors, and there are no commercialized, standardized kits available, so they have not been widely used in clinical practice. Reported enzymes include Lysyl Oxidase, Serum Immunoreactive Proline Hydroxylase (S-IR-β-PH), Plasma Prolidase, and N-acetyl-β-glucosaminidase (NAG), all of which have some significance in the diagnosis of liver fibrosis and cirrhosis. As for Monoamine Oxidase (MAO), which was previously considered an indicator of cirrhosis, its sensitivity and specificity in diagnosing early liver fibrosis are not high, and it is now rarely used.

bubble_chart Treatment Measures

Anti-fibrotic therapy aims to reduce the degree of fibrosis, delay its progression, and even reverse its pathological process. To this end, anti-fibrotic therapy should include: ① Targeting the disease cause, treating the primary disease. Such as anti-hepatitis virus, treating schistosomiasis, preventing and treating alcoholism, etc.; ② Treatment against the occurrence and development of liver fibrosis; ③ If it has developed to cirrhosis, it is necessary to prevent and treat its complications; ④ Symptomatic treatment and restoration of liver function.

Liver fibrosis is the common pathological basis for the development of cirrhosis from various disease causes. In recent years, with a deeper understanding of the mechanism of liver fibrosis, especially the molecular biology of ECM synthesis and degradation, many research efforts have proposed therapeutic measures at various stages of ECM metabolism, particularly collagen synthesis and degradation. The main ones are detailed below.

I. Inhibiting Collagen Synthesis

1. Interferon

γ-Interferon can inhibit the activation and proliferation of fat-storing cells, reduce the steady-state levels of α-Actin, type I and IV collagen, and fibronectin mRNA. There are clinical reports abroad that small doses have mild side effects and may be effective in treating liver fibrosis. α-Interferon has antiviral effects and can reduce type I collagen mRNA and TGF-β mRNA in the tissues of chronic hepatitis C patients, alleviating liver fibrosis. However, on the other hand, γ-interferon can also activate macrophages to produce IL-1 and TNF-α, which can stimulate the DNA synthesis and proliferation of fat-storing cells. This may not be beneficial for liver fibrosis. The net effect of these two aspects still requires further observation.

2. Adrenocortical Hormones

Experiments have shown that they inhibit the expression of type I collagen mRNA in cell cultures and whole animals, reducing the level of type I collagen mRNA in liver cells and fibroblasts, thereby inhibiting collagen synthesis. However, due to the many systemic side effects of long-term use, they are rarely used in anti-liver fibrosis treatment. Although they can lead to clinical remission in autoimmune chronic hepatitis, they cannot prevent the formation of fibrosis.

3. Prostaglandin Analogues

Prostaglandins have extensive biological activities. It is known that in choline-deficient rats, 16,16-Dimethyl Prostaglandin E ₂ (DEMG) can reduce fibrosis and fat deposition, possibly by inhibiting the expression of type I collagen mRNA in the liver. Other studies suggest it can increase intracellular cAMP, thereby increasing intracellular degradation. It also increases liver blood flow, changes membrane fluidity, alters blood levels of insulin and glucagon, and inhibits the release of inflammatory factors by macrophages. However, it has not yet been used for human liver fibrosis.

4. Retinoids

These drugs, such as Trans-retinoic Acid and B-cis-retinoic Acid, reduce the synthesis of type I collagen and its mRNA, and decrease the secretion of collagenase by monocytes. However, since they are also components of Ito cells, they may increase type I or III collagen, making them not necessarily suitable for treating liver fibrosis.

5. Procollagen Peptides

It is known that the terminal peptides (non-globular extensions) of procollagen can be cleaved by specific peptidases. The cleaved peptides, such as the amino-terminal peptide (PⅢP), can feedback inhibit procollagen synthesis. Before formal application, detailed molecular biology research is needed, and there are currently no reports on its use in treatment.

II. Acting on Post-Translational Steps of Procollagen mRNA

1. Colchicine

This medication has been clinically tested both domestically and internationally. Experimental studies suggest that colchicine is an anti-microtubule drug that inhibits the polymerization of tubulin, thereby interfering with collagen secretion in cells. It also stimulates the activity of collagenase, enhances degradation, acts on macrophages, inhibits monocyte factors, suppresses the release of growth factors, and reduces the secretion of interleukin I. Kershenobich and Rojkind, among others, have conducted years of observation on the use of colchicine in treating liver cirrhosis. They employed a randomized, double-blind, placebo-controlled method. Over 14 years, they followed up with 100 patients with liver cirrhosis (54 due to alcohol, 41 due to hepatitis, and 14 due to other causes). The results showed that the median survival time was 11 years for the treatment group and 3.5 years for the placebo group (P=0.0006). The 10-year cumulative survival rates were 56% and 20%, respectively (P=0.0006). In the treatment group, 30 patients underwent continuous liver biopsies, with 9 showing histological improvement, while in the placebo group, 14 patients underwent continuous liver biopsies, with none showing improvement (P=0.002). This study suggests that colchicine has a certain effect on prolonging survival rates. However, other clinical studies have not been able to confirm these results. For example, some researchers (1987, 1988) used colchicine to treat primary biliary cirrhosis and found that it did not prevent the progression of liver fibrosis. Wang Y.C. (1994) treated 100 cases of hepatitis B-related liver cirrhosis using a double-blind randomized controlled method and found no efficacy in the colchicine group compared to the control group, whether in terms of liver histological changes, serum fibrosis indicators, disease progression, or mortality rates. It seems that this drug holds little promise in anti-fibrosis treatment.

2. Proline-4-Hydroxylase Inhibitors

Experimental evidence shows that it can inhibit the hydroxylation of proline, reduce the formation of hydroxyproline, and thus decrease the stability of the triple helical α-peptide chains of procollagen. Two pyridine compounds, Pyridine 2,2-dicarboxylate (2,2-PDCA) and Pyridine 2,2-dicarboxylate, have shown anti-fibrotic effects in animal experiments, but clinical trials were discontinued due to side effects.

3. Metal Ion Chelators

Bipyridine (αα'-dipyridyl) can chelate iron ions and inhibit the activity of proline hydroxylase, causing newly synthesized collagen α-peptide chains to be released within cells due to inhibited hydroxylation, preventing the formation of triple helices. Recently, it has also been found to reduce the stability of type I collagen mRNA, thereby decreasing collagen synthesis. Its anti-fibrotic effects still require further research for confirmation. D-Penicillamine is a copper ion chelator, and since copper ions are essential cofactors for lysyl oxidase, this drug can inhibit the enzyme's activity, preventing newly secreted collagen from cross-linking. It has shown good efficacy in treating Wilson's disease but has no significant effect on liver fibrosis caused by other factors in humans and animals, and long-term use has considerable side effects.

4. Proline Analogs

These compounds, such as Azetidine Carboxylic Acid and cis-4-Hydroxyproline, can replace proline and incorporate into procollagen, forming non-helical collagen. This type of collagen is easily hydrolyzed by proteases, leading to reduced extracellular matrix formation. So far, experiments have only been conducted in rats, showing reduced collagen effects. Its toxicity is high, and it has not yet been tested in humans.

5. Inhibitors of Procollagen to Collagen Conversion

Their primary function is to prevent the cleavage of peptides at both ends of the procollagen molecule, preventing the formation of collagen molecules and impairing collagen stability. The first to be tested was arginine and its analogs (Convanine), which have not yet been used in animal experiments or human treatment.

6. Lathyrogens

These contain β-aminopropionitrile and aminoacetonitrile, which can inhibit the activity of lysyl oxidase, preventing the cross-linking of collagen fibers. Currently, research on liver fibrosis is limited to experimental studies and has not been applied to human liver fibrosis.

III. Treatments Promoting Collagen Degradation

Research on drugs that promote collagenase activity is still in its early stages. However, this class of drugs holds significant clinical importance because promoting the degradation of excessively deposited collagen could potentially reverse established liver fibrosis. Lieber C.S. et al. reported that unsaturated phosphatidylcholine (PUL) can alleviate alcoholic cirrhosis in baboons. Further in vitro cell culture studies found that it does not affect the expression of type I procollagen mRNA but doubles the collagenase activity in fat-storing cells. This suggests that promoting degradation activity may be the mechanism by which PUL treats alcoholic liver fibrosis in baboons. Multicenter clinical trials are currently underway.

IV. Gene Therapy

Wu CH et al. reported that using antisense oligonucleotide (ASO) DNA with asialoorosomucoid polylysine (AsORPL) as a carrier, targeting 3-T-AsGR cells, showed that ASOE and ASOC could inhibit type I procollagen mRNA by 73% and 67%, respectively. This work opens up prospects for gene therapy in anti-liver fibrosis.

From the above, although experimental research has been conducted on various aspects of collagen metabolism, establishing an effective treatment with minimal side effects still requires long-term efforts.

V. Treatment with Chinese Medicine and Chinese Medicinals

Since the establishment of New China, there has been significant progress in the application of traditional Chinese medicine for the treatment of chronic liver diseases. Among these, experimental and clinical research related to anti-fibrosis or the treatment of liver cirrhosis includes: Qianggan Ruanjian Decoction (Han Jinghuan, 1979), bottle gourd peel element B, oleanolic acid (Han Dewu, 1979, 1981), Liquorice Root sweet element and Liquorice Root secondary acid (Zhao Minqi et al., 1983), Salvia and other herbs (Wang Zhenling et al., 1982), Chinese caterpillar fungus mycelium, Salvia (Ma Xuehui et al., 1988), Peach Kernel and Chinese Caterpillar Fungus (Wang Yurun, Liu Ping, Liu Cheng, 1985, 1985), Tian Sanqi (Xiao Jiacheng, 1988), compound formula Salvia mixture (Wang Baoen et al., 1990), Xuefu Zhuyu Decoction (Song Jiawu, Li Shaobai, 1991), Fugankang infusion granule (Yang Zhengyun et al., 1990), tonifying the kidney and replenishing essence, tonifying qi and nourishing yin, clearing heat and removing toxin formulas (Fan Zongpang et al., 1991), Chinese caterpillar fungus mycelium (Zhang Lihuang et al., 1992), Ruangan infusion granule (Li Yanfu, 1992), etc. Recent studies have shown that 62.6% of pathologically confirmed grade I chronic hepatitis cases have liver fibrosis, while 100% of grade II, grade III chronic hepatitis, and liver cirrhosis cases have liver fibrosis. Therefore, the treatment of liver fibrosis with Chinese medicine should start from the overall pattern identification of chronic hepatitis and liver cirrhosis, based on the mechanism of disease, to establish principles, methods, prescriptions, and medications.

In the literature of traditional Chinese medicine, there was originally no disease name for chronic hepatitis and cirrhosis. Instead, they were included under the disease categories of "jaundice," "hypochondriac pain," "blood stasis," "abdominal masses," and "tympanites." Modern research in Chinese medicine suggests that chronic hepatitis and early cirrhosis are caused by lingering dampness-heat pathogens that have not been cleared, which over time damage the zang-fu organs, qi, and blood. Due to the pathogens lurking in the blood aspect and the deficiency of healthy qi, liver qi becomes depressed, leading to blood stasis and stagnation. When healthy qi fails to circulate, phlegm turbidity congeals, and qi deficiency combined with blood stasis contributes to the formation of abdominal masses. Prolonged liver disease affects the spleen and stomach, disrupting the ascending and descending functions, weakening qi movement, and further exacerbating blood deficiency and qi deficiency. Over time, the disease may also involve the kidneys, leading to liver-kidney yin deficiency or spleen-kidney yang deficiency. As a result, the liver, spleen, and kidneys are all impaired, and qi, blood, and water become entangled. Some Chinese medicine experts point out that the central mechanism of the disease is: qi deficiency and blood stasis are the root of cirrhosis, while the retention of dampness-toxin and pathogenic heat in the blood aspect is the manifestation. The liver lacks blood nourishment and loses its softness, while the kidneys lack clear nourishment and lose their moisture. The treatment principle should focus on activating blood, tonifying qi, nourishing blood, and pacifying the liver, supplemented by nourishing water to moisten wood or warming and tonifying the spleen and kidneys. If residual pathogens remain, they should be expelled.

Based on the above, recent treatments for chronic hepatitis and early cirrhosis in Chinese medicine have primarily focused on the principles of activating blood and resolving stasis, combined with tonifying qi, tonifying deficiency, nourishing blood, pacifying the liver, or nourishing water to moisten wood.

Through a series of experimental studies, Ma Xuehui, Han Dewu, and others have shown that Salvia can reduce liver cell degeneration and necrosis, lower alanine aminotransferase levels, reduce lipids, and increase liver glycogen in animal models of acute liver injury. In animals with partial liver resection, it promotes liver cell regeneration. In experimental liver fibrosis, it prevents the occurrence of cirrhosis and promotes the reabsorption of collagen in the liver. It also has preventive and therapeutic effects on experimental acute liver failure. The mechanism may involve reducing endogenous toxinemia, enhancing Kupffer cell function, preventing free radical production, reducing lipid peroxidation, stabilizing liver cell membranes and organelle membranes, increasing liver drug enzymes, and preventing Ca⁺⁺ influx. In experimental liver fibrosis and cirrhosis, Salvia has been observed to reduce serum hyaluronic acid levels, decrease collagen content in the liver, and reduce the production of Fn and Ln.

Wang Yurun, Liu Ping, Liu Cheng, and others used Peach Kernel extract combined with Chinese caterpillar fungus mycelium to treat schistosomiasis-induced liver fibrosis. Using PⅢP, blood and urine hydroxyproline, and liver tissue collagenase as observation indicators, the results showed that after the pathogen (i.e., schistosome) was treated, this treatment had a certain promoting effect on the catabolism of collagen in the liver of patients with liver fibrosis.

This chapter discusses the observation and treatment of experimental liver fibrosis using a compound formula of Chinese medicinals primarily composed of Salvia, as studied by Wang Baoen et al. It was observed that the invigorating blood and resolving stasis Chinese medicinals have preventive and therapeutic effects on CCL₄ toxic injury and albumin immune injury-induced liver fibrosis. The application of this medicine can reduce the progression of liver fibrosis to varying degrees and can also significantly reverse fibrosis in those who have already developed liver cirrhosis. Pathohistological observations indicate that the compound formula for invigorating blood and resolving stasis can inhibit the proliferation and deposition of type I, III, and V collagen in fibrous connective tissue, preventing its extension along and into the lobules. Meanwhile, laboratory studies have also observed that this compound formula, whether used preventively or therapeutically, significantly increases the activity of collagenase in the liver tissue and serum of rats with liver fibrosis, and can increase the ratio of active collagenase to latent collagenase activity in liver tissue. This indicates that the increase in degradation activity is another important aspect of the anti-fibrosis mechanism of invigorating blood and resolving stasis Chinese medicinals. As for the site of action of this Chinese medicinals compound formula, molecular biology studies have shown that it can inhibit the expression of type II and III collagen mRNA in liver tissue and can inhibit the expression of type IV collagen mRNA in cultured fat-storing cells in vitro. This suggests that the site of action of Chinese medicinals is at the transcriptional level of collagen genes. Clinical applications in patients with chronic hepatitis and liver cirrhosis have also shown good results. Manifestations include the disappearance or alleviation of symptoms after treatment, reduction in spleen size, and alleviation of portal vein diameter widening. Seventy-three percent of those with elevated transaminase levels returned to normal. The levels of serum PIII P and Ln, which were elevated, returned to normal. This suggests that this formula is an effective anti-fibrosis formula.

In addition to the principle of promoting blood circulation and removing blood stasis, there are also experimental studies on other principles that have shown therapeutic effects, such as the use of Chinese medicinals based on the principles of tonifying the kidney and replenishing essence, tonifying qi and nourishing yin, and clearing heat and removing toxins, all of which have varying degrees of therapeutic effects on liver fibrosis in rats.

From a large amount of domestic work, it appears that Chinese medicine and Chinese medicinals have great potential in treating liver fibrosis and should be given due attention.