Application of subcellular extract of stem cells in preparation of medicine for treating liver injury

Abstract

The invention relates to stem cells, in particular to application of a subcellular extract of stem cells in preparation of a medicine for treating liver injury. Experiments prove that the prepared mesenchymal stem cell subcellular extract has a remarkable improvement effect on a carbon tetrachloride induced mouse hepatic fibrosis model in vivo and in vitro. The stem cell subcellular extract prepared by a mechanical disruption method can effectively alleviate the morphological problems such as hepatomegaly and rough surface, and improve the symptoms of hepatocyte injury and collagen deposition. In addition, the subcellular extract of the stem cells can significantly reduce the serum transaminase level of mice with hepatic fibrosis and improve the liver function. Meanwhile, by inhibiting the expression of fibrosis-related genes and inflammation-related genes in the liver, the subcellular extract of the stem cells exerts remarkable anti-fibrosis and anti-inflammatory effects. The umbilical cord mesenchymal stem cell subcell extract prepared by a mechanical crushing process provides a new method and thought for clinical treatment of liver injury.

Description

Technical Field

[0001] The invention belongs to the field of medical biotechnology and relates to the application of a subcellular extract of stem cells in the preparation of a medicine for treating liver damage. Background Art

[0002] The number of patients with liver diseases is increasing day by day, and it has become a global public health problem. The causes of liver damage and liver disease are very wide, and alcohol, drugs and hepatitis viruses can cause acute or chronic liver damage, leading to liver fibrosis, cirrhosis and liver failure.

[0003] For patients with end-stage liver disease, the only option to improve their survival rate is liver transplantation. However, liver transplantation faces many problems, such as severe shortage of liver donors, high cost, high surgical risk, risk of postoperative complications and postoperative immune rejection, which ultimately lead to the loss of treatment opportunities for patients with end-stage chronic liver disease and increased mortality. Therefore, there is an urgent need to find effective treatments to replace liver transplantation.

[0004] In recent years, with the rise of cell therapy, researchers have discovered that stem cells are rich in sources and have low immunogenicity, and thus are widely used in the treatment of a variety of diseases. Among them, stem cells can improve the clinical symptoms of patients with liver disease, reduce the incidence and mortality of cirrhosis / liver cancer, and are increasingly becoming a new idea for the treatment of liver diseases. Stem cells themselves are expensive, so exploring stem cell active ingredient extracts as by-products has great practical significance. Currently, a variety of stem cell active ingredient extracts have been discovered, and these extracts have also been proven to be druggable in research.

[0005] Therefore, the present invention prepares a subcellular extract of mesenchymal stem cells, verifies the repair effect of the extract on liver damage in in vivo experiments, investigates the possibility and feasibility of using stem cell subcellular extract as its effective ingredient to treat liver damage, and promotes the secondary development of mesenchymal stem cell drugs. Summary of the invention

[0006] Purpose of the Invention

[0007] The purpose of the present invention is to provide a stem cell subcellular extract as an effective ingredient, a preparation method thereof and an application thereof in treating liver damage.

[0008] Technical Solution

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] A use of a subcellular extract of stem cells in the preparation of a drug for treating liver damage, characterized in that the subcellular extract of stem cells is prepared by a crushing method to prepare a stem cell lysate, and the subcellular extract is obtained by centrifuging a precipitate.

[0011] The application is characterized in that the stem cells include umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, skin mesenchymal stem cells, bone marrow mesenchymal stem cells or amniotic membrane mesenchymal stem cells.

[0012] The stem cell subcellular extract of the present invention contains mesenchymal stem cells, especially mesenchymal stem cells obtained from the umbilical cord. The stem cells are primary to seventh generation mesenchymal stem cells.

[0013] A stem cell subcellular extract for treating liver damage, wherein the subcellular extract, before extraction, comprises: a mesenchymal stem cell suspension, wherein the density of the mesenchymal stem cells in the mesenchymal stem cell suspension is 2×10 6 ~1×10 7 The mesenchymal stem cell suspension was prepared with sterile PBS buffer and the cells were evenly distributed in the suspension.

[0014] Preferably, the mesenchymal stem cell suspension is homogenized 100 times in an ice bath by a glass homogenizer to prepare a mechanically disrupted lysate of the stem cells, and observed under a microscope, and the cell viability is determined to be below 1% by trypan blue staining. If the viability is greater than 1%, it is re-transferred into the glass homogenizer and ground 20 times in an ice bath until the cell viability meets the requirements.

[0015] Preferably, the stem cell lysate suspension is transferred into a centrifuge tube, centrifuged at 2000 rpm for 3 minutes, the supernatant is discarded, and then washed once with sterile PBS, and finally the precipitate is fully mixed with PBS to prepare a stem cell subcellular extract (MSC-CD) suspension.

[0016] The medicine for treating liver damage of the present invention comprises the prepared stem cell subcellular extract and pharmaceutically acceptable excipients.

[0017] Furthermore, the pharmaceutically acceptable excipients include, but are not limited to, conventional pharmaceutically acceptable penetration enhancers, absorption enhancers, osmotic pressure regulators, surfactants, pH regulators, antioxidants, stabilizers, fillers, adhesives, excipients, chelating agents, propellants, pore formers, film formers, solvents, cosolvents, etc.

[0018] Another object of the present invention is to provide the use of the stem cell subcellular extract (MSC-CD) in treating liver damage.

[0019] Preferably, the application is the application of the stem cell subcellular extract in the preparation of a drug for repairing liver damage.

[0020] Preferably, the liver damage includes but is not limited to acute or chronic liver damage caused by alcoholic or non-alcoholic factors, including liver inflammation, liver fibrosis, cirrhosis, liver failure or liver cancer.

[0021] Furthermore, in the present invention, we used the prepared stem cell subcellular extracts to treat mice with chronic liver fibrosis that had been successfully established by intraperitoneal injection of carbon tetrachloride, and evaluated the improvement effect on their liver damage.

[0022] The key technical points of the present invention are:

[0023] (1) Subcellular components of mesenchymal stem cells were extracted, and an unreported method for extracting subcellular components of stem cells by mechanical disruption was provided.

[0024] (2) It was demonstrated that the prepared stem cell subcellular extract had an ameliorative effect on liver lesions, significantly improving collagen production and liver cell damage.

[0025] (3) It was shown that stem cell subcellular extracts have anti-inflammatory effects.

[0026] Beneficial Effects

[0027] (1) The stem cell subcellular extract prepared by the present invention has excellent anti-fibrosis and liver damage repair effects. Although mesenchymal stem cells have been proven to have anti-fibrosis effects, stem cells can be induced to differentiate into lung cells under specific conditions, and have the effects of "homing", differentiation, secretion of various bioactive substances to participate in regulating immune responses, etc., and their mechanisms of action include: Antioxidant mechanism: Mesenchymal stem cells have certain antioxidant and oxygen free radical scavenging effects, which help to alleviate pulmonary fibrosis. Cell fusion, cell-cell interaction, differentiation, and promotion of angiogenesis: These mechanisms, either alone or in interaction, will play an anti-fibrotic role. Anti-inflammatory and immunomodulatory mechanism: Mesenchymal stem cells exert anti-inflammatory immunomodulatory effects by secreting anti-inflammatory factors and antibacterial peptides or inhibiting pro-inflammatory signaling pathways, upregulating inflammatory signaling pathways and bacterial phagocytosis, etc., and alleviate pulmonary fibrosis. Paracrine mechanism: Mesenchymal stem cells secrete many factors, including hepatocyte growth factor, angiopoietin-1, vascular endothelial growth factor, certain chemokines, inflammatory factors and regulatory peptides, etc., which directly or indirectly alleviate inflammatory responses and pulmonary fibrosis in the lungs.

[0028] The above mentioned effects are all produced by living stem cells, but for the present invention, they are subcellular extracts, which are obtained by removing the supernatant from the stem cell lysate suspension. In fact, the soluble stem cell secretory factors are removed to obtain subcellular fragments.

[0029] There are no literature reports on the effects of subcellular extracts in combating liver fibrosis and repairing liver damage. The inventors of the present application systematically examined the therapeutic effects of the stem cell subcellular extracts on chronic liver damage in vivo and in vitro, and verified that the extracts can significantly improve liver swelling, promote damage repair, reduce collagen formation, and improve liver inflammation. The present invention hopes to promote the research and development of stem cell active ingredient extracts, and provide an effective new option for stem cell medicine and the treatment of liver damage.

[0030] (2) The extraction process for preparing stem cell subcellular extracts of the present invention has simple steps, low raw material cost, simple and fast operation, and can obtain stable raw materials through freeze-drying process.

[0031] (3) The present invention established a mouse model of chronic liver fibrosis and found that the prepared stem cell subcellular extract can improve liver fibrosis and liver damage in vivo and in vitro, significantly reduce collagen deposition (p < 0.001), and significantly reduce serum alanine aminotransferase (p < 0.0001) and aspartate aminotransferase (p < 0.0001); and studied the downregulation of genes related to liver fibrosis (p < 0.001) and inflammation (p < 0.05) in mice with chronic liver damage by the stem cell subcellular extract. It is suggested that the prepared stem cell subcellular extract has a significant effect in the treatment of chronic liver damage, and further proposed that the extract plays a therapeutic effect as an effective component of stem cell treatment of liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Relative mRNA expression levels of fibrosis genes Acta2 (A), Col1a2 (B), Col3a1 (C), and Desmin (D) in the primary non-parenchymal cell population of the liver of mice with chronic liver injury after administration of in vitro stem cell subcellular extracts.

[0033] Figure 2 The results of anatomical observation of the liver of each group of mice after carbon tetrachloride modeling and administration (A) and the liver index (liver weight / body weight ratio) of each group of mice (B).

[0034] Figure 3 The picrosirius red staining (A), H&E staining (B), α-SMA immunohistochemistry (C), and quantitative statistics of the picrosirius red positive area (D) and α-SMA positive area (E) of the liver of each group of mice.

[0035] Figure 4 Figure 2 shows the levels of aspartate aminotransferase (A) and alanine aminotransferase (B) in the serum of mice in each group.

[0036] Figure 5 The contents of α-SMA (A) and hydroxyproline (B) in liver tissue homogenate of mice in each group.

[0037] Figure 6 The relative mRNA expression levels of the fibrosis genes Acta2 (A), Col1a2 (B), Col3a1 (C), and Desmin (D) in the liver of each group of mice.

[0038] Figure 7 Relative mRNA expression levels of M1 pro-inflammatory macrophage genes CCL2 (A) and IL-6 (B) in the liver of mice in each group. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with the accompanying drawings. The embodiments described below are only some embodiments of the present invention, not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The present invention designs and successfully prepares a stem cell subcellular extract as an injection, wherein the main active ingredient of the injection is a mesenchymal stem cell subcellular extract collected by mechanical crushing and centrifugation.

[0041] The experimental methods without specific conditions and reagents without formulas in the examples are all conventional conditions in the art.

[0042] As a preferred embodiment, the present invention also provides a method for preparing a stem cell subcellular extract for treating liver damage, the steps of which include:

[0043] Step (I): Under a sterile operating environment, mesenchymal stem cells were placed in a standard incubator at 37°C and 5% CO2 and cultured with complete F12 / DMEM medium. The cells were passaged every 3 days, and primary to 7th generation mesenchymal stem cells in good condition were selected for subsequent operations.

[0044] Step (ii): Digest the cells with 0.1% trypsin, count them and resuspend them in sterile PBS buffer to a density of 2×10 6 ~1×10 7 indivual.

[0045] Step (III): Transfer the stem cell suspension to a sterile handheld glass homogenizer, grind 100 times in an ice bath, stain with trypan blue and observe under a microscope to determine if the cell viability is below 1%. If the viability is greater than 1%, transfer it to the glass homogenizer again and grind it 20 times in an ice bath until a cell fragment suspension with a cell viability that meets the requirements is obtained.

[0046] Step (iv): transfer the cell fragment suspension into a centrifuge tube, place it in a high-speed centrifuge, centrifuge at 2000 rpm for 3 minutes, discard the supernatant, wash it with sterile PBS, and finally mix the precipitate with physiological saline to prepare a stem cell subcellular extract suspension.

[0047] Example 1: In vitro anti-fibrotic efficacy of stem cell subcellular extracts on primary hepatocytes from mice with chronic liver injury

[0048] Establishment of CCl4 chronic liver injury mouse model: C57BL / 6N mice that had been adapted for one week were randomly divided into a normal control group and a carbon tetrachloride modeling group. The mice in the carbon tetrachloride modeling group were intraperitoneally injected with 20% carbon tetrachloride, 5mL / Kg, 3 times a week for 8 weeks. During this period, the mice in the normal control group were also intraperitoneally injected with solvent soybean oil. Primary liver cells were extracted at the 9th week.

[0049] Extraction of mouse primary liver cells: A two-step perfusion method was used to extract and separate mouse primary liver cells, and the whole process was aseptically operated in a biological safety cabinet. The mice in the normal control group and the carbon tetrachloride modeling group were anesthetized with 1% sodium pentobarbital. After disinfecting the abdomen of the mice with 75% ethanol, the mice were fixed on a sterile operating table, the abdominal cavity was opened, and the portal vein and inferior vena cava of the mice were found and exposed. The perfusion tube was inserted into the inferior vena cava and fixed with sutures; the superior vena aorta was ligated with an artery clamp, and then the portal vein was cut to form a single pathway for the perfusion fluid. The perfusion pump then perfused the liver with 37°C liver perfusion buffer at a uniform speed, and the perfusion was stopped when the liver color remained khaki. Replace it with 37°C preheated type IV collagenase digestion solution, reduce the flow rate and perfuse at a uniform speed, and press the liver during perfusion to fully digest the cells. The digested liver was removed and placed in a sterile culture dish containing a small amount of collagenase, and placed on ice for later use.

[0050] Isolation of primary non-parenchymal cells of the liver: Add complete blank culture medium to the digested liver tissue to stop digestion. Use sterile tweezers to break the liver capsule to release the dispersed cells into the culture dish; then pass through a 100-mesh sterile cell sieve to obtain a cell suspension, and place the filtered cell suspension into a 50 mL sterile centrifuge tube. Add culture medium to make up the volume to 50 mL, centrifuge for 2 minutes (4°C, 50g), keep the supernatant, and discard the precipitate. Repeat this washing twice and collect the cell supernatant. Combine all supernatants and centrifuge for 15 minutes (4°C, 1350g), discard the supernatant, and you will get the primary non-parenchymal cells (pNPCs) of the liver.

[0051] Culture of primary liver non-parenchymal cells and administration of stem cell subcellular extracts: pNPCs were spotted into the cell plate and cultured with complete DMEM medium. The medium was changed after 2 hours. After 12 hours, when the cells were completely attached and fused, MSC-CD prepared by adding pNPCs:MSC=100:1 was added. After interacting with pHSCs for 48 hours, pHSCs were collected and RNA was extracted by Trizol method. After reverse transcription into cDNA, q-PCR gene quantitative determination was performed. The results are shown in Figure 1 shown.

[0052] Figure 1 The results showed that the prepared stem cell subcellular extracts had a significant inhibitory effect on the activation of fibrotic genes in primary liver non-parenchymal cell populations in vitro. Acta2, Col1a2, Col3a1, and Desmin play an important role in the fibrosis process, and their expression levels and activities are usually positively correlated with the degree of fibrosis. Figure 1 AD showed that compared with the normal control group, Acta2, Col1a2, Col3a1, and Desmin in the carbon tetrachloride modeling group were significantly upregulated, while after administration of stem cell subcellular extracts, the above genes were significantly reduced (one-way analysis of variance, P<0.01). That is, the prepared stem cell subcellular extract has a significant anti-liver fibrosis effect in vitro.

[0053] Example 2: In vivo therapeutic efficacy of stem cell subcellular extracts on mice with chronic liver injury.

[0054] Administration of stem cell subcellular extracts to mice with CCl4 chronic liver injury: The same chronic liver injury model was established in C57BL / 6N mice as described in Example 1. However, 7 mice from the carbon tetrachloride modeling group were further divided into the administration group. The stem cell subcellular extracts were injected into the tail vein starting from the 6th week of carbon tetrachloride modeling. Sterile saline was used as the solvent, and the dose was 5×10 5 The subcellular extract of stem cells was injected once a week for 3 consecutive weeks. During this period, the carbon tetrachloride modeling group and the extract treatment group were continuously given carbon tetrachloride intraperitoneal injection three times a week to maintain the chronic liver injury model.

[0055] Animal sample collection: One week after the last administration, mice were fasted but not watered for 12 hours, anesthetized with 1% sodium pentobarbital and weighed. Mice were killed after orbital blood was collected, and the liver was taken and weighed. Part of the liver tissue was cut and fixed in 4% paraformaldehyde fixative for the preparation of paraffin sections, and the rest of the liver tissue was stored in a -80°C refrigerator for later use.

[0056] Liver tissue morphology observation: Liver samples were fixed with 4% paraformaldehyde, embedded in paraffin and sliced ​​(5μm). Then the liver sections were stained with hematoxylin and eosin (H&E): first, the nuclei were stained with hematoxylin for 5 minutes, then washed with tap water 5 times to wash off the dye, then hydrochloric acid alcohol tissue was differentiated for 30 seconds, rinsed with running water, and finally stained with 1% eosin cytoplasm for 1 minute. The stained sections were soaked in 95% ethanol and anhydrous ethanol twice, each for 1 minute to dehydrate; soaked in xylene 3 times, each for 2 minutes to make it transparent; finally, the sections were sealed with neutral gum. After the paraffin sections were dehydrated, they were stained with Sirius red staining solution for 8 minutes, rinsed in anhydrous alcohol for several minutes, and observed under a microscope. After the sections were dried in an oven at 60℃, they were transparentized with xylene for 5 minutes and sealed with neutral gum. Under an optical microscope, collagen fibers appeared red and the background was yellow. Each section was observed with a fluorescent inverted microscope, and 3 fields of view were randomly selected for each section for photography (200×).

[0057] Separation of serum and determination of transaminase: After the collected mouse whole blood was left at room temperature for 3 hours, it was centrifuged at 8000 rpm for 5 minutes at room temperature, and the supernatant was collected and frozen in a -80°C refrigerator for later use. Serum aspartate aminotransferase and alanine aminotransferase were detected using ELISA kits, and all operations were carried out strictly in accordance with the instructions.

[0058] Determination of liver-related indicators: 30 mg of liver tissue was taken, 270 μL of normal saline was added, 2-3 beads were added, and the tissue was ground by a tissue homogenizer until the tissue block disappeared completely. After centrifugation at 14000g for 10 minutes, the supernatant was taken and frozen in a -80℃ refrigerator for later use. The liver homogenate was tested for the content of α-SMA and hydroxyproline in the liver using an ELISA detection kit, and all operations were carried out strictly in accordance with the instructions.

[0059] Determination of mRNA expression of liver-related genes: 30 mg of liver lobe was taken, 1 mL of Trizol lysis solution was added, and the liver total RNA was extracted using a handheld homogenizer until there was no lumpy tissue. After reverse transcription into cDNA, q-PCR quantitative determination was performed to detect the mRNA expression of fibrosis-related genes (Acta2, Col1a2, Col3a1, desmin) and inflammation-related genes (CCL2, IL-6) in the liver.

[0060] The results are as follows Figure 2 As shown, Figure 2 (A) shows that stem cell subcellular extracts have a significant improvement effect on the appearance of the liver in chronic liver damage, effectively reducing morphological problems such as liver enlargement and surface roughness. Figure 2(B) shows that after 9 weeks of chronic liver injury modeling, the liver index (liver weight / body weight ratio) of mice in the carbon tetrachloride modeling group was significantly increased compared with that in the normal control group (one-way analysis of variance, p<0.001), while after three treatments with stem cell subcellular extracts, the liver index of mice was significantly decreased (one-way analysis of variance, p<0.05). Figure 3 The results of H&E staining showed that the carbon tetrachloride modeling group had focal lesions around the central vein, moderate or severe hepatocyte edema, fatty degeneration and necrosis, and a large number of immune cell infiltration, which were significantly relieved after treatment with stem cell subcellular extracts. The hepatocytes in the extract-treated group were neatly arranged, similar to the liver sections of the normal control group. The results of immunohistochemical staining of α-SMA also showed that the α-SMA positive area increased significantly after carbon tetrachloride modeling, and the α-SMA positive area decreased after extract treatment, and the positive area area was also statistically different (one-way analysis of variance, p<0.0001). Figure 4 The results showed that after 9 weeks of chronic liver injury modeling, the serum transaminases aspartate aminotransferase and alanine aminotransferase in the carbon tetrachloride modeling group were significantly increased compared with the normal control group, and were significantly restored after subcellular extract treatment (one-way analysis of variance, p<0.0001). Figure 5 The results showed that α-SMA and hydroxyproline in the liver of mice were significantly increased after modeling, and significantly decreased after treatment with subcellular extracts (one-way analysis of variance, p<0.05). Figure 6 The results showed that compared with the normal control group, the fibrosis-related genes Acta2, Col1a2, Col3a1, and Desmin in the carbon tetrachloride modeling group were significantly upregulated, while after the administration of subcellular extracts, the above genes were significantly decreased (one-way analysis of variance, P<0.001). Figure 7 To detect inflammation-related indicators, Figure 7 The results showed that the mRNA expression of pro-inflammatory M1 macrophage markers CCL2 (A) and IL-6 (B) in the liver increased after modeling, while decreased after administration of subcellular extracts (one-way ANOVA, p<0.05), indicating that stem cell subcellular extracts have anti-inflammatory effects.

[0061] Therefore, this study shows that the prepared stem cell subcellular extract has the same results in in vivo experiments as in in vitro experiments, and has a significant improvement effect on chronic liver injury and liver fibrosis, can reduce liver damage, reduce liver index, and improve the inflammatory environment of macrophage infiltration.

[0062] Therefore, this study shows that the prepared stem cell subcellular extract has a significant improvement effect on chronic liver injury, can reduce liver damage, inhibit inflammatory response and alleviate fibrosis. These results provide a strong experimental basis for the potential application of the prepared stem cell subcellular extract in the treatment of liver injury.

[0063] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all details in detail, nor do they limit the invention to only the specific implementation methods described. According to the content of this specification, many modifications and changes can be made. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and use the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Use of a subcellular extract of stem cells in the preparation of a drug for treating liver damage, characterized in that: The subcellular extract of the stem cells is prepared by a disruption method to prepare a stem cell lysate, and the subcellular extract is obtained by centrifuging a precipitate.

2. The use according to claim 1, characterized in that: The stem cells include umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, skin mesenchymal stem cells, bone marrow mesenchymal stem cells or amniotic membrane mesenchymal stem cells.

3. The use according to claim 1, characterized in that: The stem cell subcellular extract is prepared according to the following steps: Step (1), preparation of mesenchymal stem cell suspension: Under a sterile environment, primary to 7th generation mesenchymal stem cells in good condition were taken, digested with 0.1% trypsin, counted, and resuspended in sterile PBS buffer to a density of 2×10 mesenchymal stem cells in the suspension. 6 ~1×10 7 Pieces / mL; Step (2), disruption of mesenchymal stem cells: the cell suspension was transferred to a sterile glass homogenizer, ground 100 times in an ice bath, and the cell viability was determined to be below 1% by trypan blue staining; if the viability was greater than 1%, the suspension was re-transferred to a glass homogenizer and ground 20 times in an ice bath until the cell viability met the requirement; Step (3), extraction of mesenchymal stem cell subcellular extracts: the cell fragment suspension was transferred into a centrifuge tube, centrifuged at 2000 rpm for 3 minutes, the supernatant was discarded, and then washed once with sterile PBS, and finally mixed thoroughly with PBS to prepare a subcellular extract suspension.

4. The use according to claim 1, characterized in that: The subcellular extract of stem cells is a complex formed by the subcellular extract of stem cells and pharmaceutically acceptable excipients.

5. The use according to any one of claims 1 to 4, characterized in that: The liver injury is acute or chronic liver injury, including liver inflammation, liver fibrosis, cirrhosis, liver failure or liver cancer.

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