End-stage liver disease (ESLD) is characterized by liver function not meeting the normal physiological needs of the human body. It is an advanced stage caused by various acute and chronic liver damage, mainly including decompensated cirrhosis and liver failure caused by diseases such as viral hepatitis , alcoholic liver disease , autoimmune hepatitis and other diseases. This type of disease has a high mortality rate and a heavy medical burden, which seriously endangers people's health. As an effective clinical treatment method, liver transplantation has problems such as shortage of donors and high costs [1]. In recent years, cell therapy research has highlighted the potential clinical application advantages of mesenchymal stem cells (MSC) in the treatment of liver disease . MSCs can be isolated from tissues such as bone marrow, fat, umbilical cord blood, and embryos, but the biology characteristics of MSCs from different sources are different. One study, [2], showed that bone marrow-derived MSCs have the strongest migration ability, which is an indispensable factor in the process of tissue regeneration and repair. At the same time, bone marrow mesenchymal stem cells (BMSCs) have strong paracrine functions, especially the exosomes from which they are derived have attracted much attention in the treatment of liver disease. This article reviews the research progress of BMSC and its exosomes in ESLD, providing some reference for further clinical application research.
1BMSC and its exosomes overview
BMSC is an adult stem cell derived from the mesoderm. It has the potential of self-renewal and multidirectional differentiation. It can be cultured and amplified in vitro and can be differentiated into a variety of histiocytic cells under specific conditions, such as bone, cartilage, cardiomyocytes, epithelial cells , etc. Currently, BMSC still lacks specific markers, which are generally believed to express CD73, CD90, and CD105, but do not express CD14, CD34, and CD45. Because of its low immunogenicity and is not easy to trigger the receptor immune response, BMSC is suitable for allogeneic or autologous transplantation. BMSCs can migrate to damaged liver tissue and differentiate directly into hepatocytes, and can also paracrine soluble factors such as prostaglandin E2, hepatocyte growth factor (HGF), indoleamine 2,3-dioxygenase and vascular endothelial growth factor to improve the microenvironment of liver tissue and promote the repair of damaged liver cells [3-4].
exosomes are extracellular vesicles that fuse with cell membrane and release into the extracellular space. The diameter is 40~160 nm. It is a new non-cellular way of signaling to transmit . Exosomes carry a large number of biologically active substances, including protein , lipid , DNA, mRNA, and microRNA. The induced changes in activity of target cells through different signaling pathways . Exosomes can be secreted and absorbed by almost all cell types, and BMSC is its important cellular source [5]. Compared with BMSC, BMSC-derived exosomes (BMSC-Ex) are easier to obtain, have no immune rejection, are safer, and have cross-biological barrier capabilities, and can be developed as a drug delivery vehicle. BMSC-Ex transmits biological information to damaged livers and participates in the regulation of pathophysiological processes such as liver cell regeneration, liver fibrosis and liver inflammation damage [6-7]. Clinical application of
2BMSC transplantation for the treatment of ESLD
has shown that BMSC transplantation has significant clinical effects on ESLD represented by decompensated cirrhosis and liver failure.
2.1 Clinical study on the treatment of decompensated cirrhosis
Decompensated cirrhosis is the end stage of chronic liver disease. Due to extensive hepatocyte necrosis and damage to liver tissue structure, liver function decompensation has occurred, and a series of serious complications have occurred [8]. BMSC transplantation has good efficacy on decompensated cirrhosis caused by various causes. A clinical trial, [9], recruited 56 patients with hepatitis B cirrhosis, and autologous BMSC was injected through the hepatic artery. Follow-up for 24 weeks, a total of 39 patients completed the trial, including 20 in the transplant group and 19 in the control group. The results showed that the improvement of liver function, end-stage liver disease model (MELD) scores and Child-Pugh scores in the autologous BMSC transplant group was significantly better than that in the conventional treatment group. Similarly, autologous BMSC was treated with hepatitis C cirrhosis [10] through peripheral venous transplantation, and alcohol cirrhosis [11] through hepatic artery transplantation can all improve patients' liver function and reduce the degree of fibrosis . However, not all studies have shown that BMSC transplantation has an ideal therapeutic effect on cirrhosis and patients.For example, a randomized controlled clinical study, [12] recruited 27 patients with cirrhosis decompensated , of which 15 patients injected autologous BMSC from peripheral intravenously, and 12 patients injected equal volumes of normal saline as control. After 12 months, there was no significant difference in the changes in liver function, Child-Pugh score, MELD score, and international standardized ratio (INR) between the two groups. This may be related to factors such as the number of cells transplanted by BMSC, the transplant pathway, and whether it is pretreated before transplantation.
2.2 Clinical study on the treatment of liver failure
Liver failure is a serious liver injury caused by various factors, leading to serious liver dysfunction, and a group of clinical syndromes mainly manifested in jaundice , ascites, coagulation dysfunction, hepatic encephalopathy, etc. A randomized controlled clinical trial of [13] was treated with patients with hepatitis B-related liver failure once a week for 4 weeks and followed up for 24 weeks. The results showed that the cumulative survival rate of the BMSC transplant group (73.2%) was significantly higher than that of the conventional treatment group (55.6%), and BMSC transplantation can significantly reduce the patient's serum TBil level, improve MELD scores, and reduce the incidence of severe infections, and improve the survival rate of 24 weeks. There were also studies that [14] pretreat BMSC and inject hepatitis C -related liver failure patients were observed in vivo. After 5 days of subcutaneous injection of granulocyte colony stimulating factor, BMSC was extracted, and after isolation and purification, it was injected into autologously intravenously in the peripheral vein for treatment; after follow-up of more than 26 weeks, serum ALT and AST in 54% of the patients in the transplant group returned to normal, and liver synthesis function improved. Another study, [15], transplanted autologous BMSC into hepatocyte-like cells and followed up for 6 months. The results showed that BMSC transplantation can significantly improve ascites, lower limb edema, serum albumin content and Child-Pugh scores in patients with liver failure.
35BMSC transplantation treatment mechanism of ESLD
ESLD pathology is characterized by inflammation and necrosis of large amounts of liver tissue and collagen fiber deposition. Intervention studies on severe liver injury and liver fibrosis models have shown that BMSC transplantation therapy can promote hepatocyte regeneration, reduce liver inflammation and inhibit the progression of liver fibrosis (Figure 1).
Figure 1 The mechanism of action of BMSC and its exosomes in the treatment of ESLD
3.1 BMSC promotes hepatocyte regeneration
Hepatocytes are the main component of liver parenchymal cells and are responsible for liver biosynthesis, transformation, metabolism and other functions, and maintain liver homeostasis. Continuous chronic liver damage causes a large number of hepatocytes to necrosis and apoptosis, and the liver cell proliferation ability is seriously insufficient and cannot meet the body's liver function needs. Therefore, effectively promoting hepatocyte regeneration is an important way to treat ESLD. BMSC transplant therapy promotes liver regeneration. If BMSC is injected into D-galactosamine/ lipopolysaccharide [16] and 90% hepatic resection [17], the positive staining area of nuclear antigen (PCNA) and Ki-67 in liver tissue proliferating cells is significantly increased, and β-catenin in hepatocytes undergoes nuclear translocation, upregulating the expression of proto-oncogene and cyclin D1 (cyclin D1) and promoting hepatocyte proliferation. This phenomenon is related to the homing of BMSCs to the damaged liver, promoting hepatocyte regeneration through direct differentiation or paracrine function. BMSC was injected into the tail vein of mice [18] of in CCl4-induced liver fibrosis, and the BMSCs marked by green fluorescent protein migrated to the damaged liver, and the expression of the hepatic cell marker Alb in the liver tissue was increased. Under the action of cytokine including HGF, vascular endothelial growth factor, galactose gluten-3 or macrophage colony stimulating factor , the ability of BMSC to honest the liver and differentiate into hepatocyte-like cells is enhanced. The mechanism of BMSC homing to the liver has not been fully elucidated, and the study of [21] showed that it was related to SDF-1/CXCR4 axis activation. During liver injury, SDF-1 expression in liver tissue is increased, stimulating the upregulation of its receptor CXCR4 on the surface of BMSC, thereby mediating the migration of BMSC to damaged liver tissue.
exosomes are an important part of the BMSC paracrine mechanism and play a biological function similar to their parent cells. Animal experiments [22] showed that BMSC-Ex could migrate to the liver of mice induced by tarsin-A (Con-A), inhibiting hepatic cell apoptosis, and promoting liver tissue regeneration and repair.Cell experiments [23] confirmed that BMSC-Ex inhibits hepatocyte apoptosis through the autophagy pathway, significantly reducing the expression of the pro-apoptotic protein Bax and cleaved caspase-3, while upregulating the expression level of the anti-apoptotic protein Bcl-2. At the same time, it can induce the G0 phase hepatocytes to re-enter the G1 phase in the hepatocyte injury model, significantly upregulating the expression of cyclin D1 and PCNA, and accelerating the hepatocyte regeneration cycle [24].
3.2 BMSC inhibits the progression of liver fibrosis
Liver fibrosis is a pathological change in response to chronic liver injury and is an important pathological basis for decompensated cirrhosis. Liver damage stimulates the activation of hepatic stellate cells (HSCs) and transdifferentiates into myofibroblasts expressing α-SMA, secreting a large amount of collagen, resulting in excessive deposition of extracellular matrix [8]. Animal experiments [25] showed that BMSC transplantation can alleviate the expression of type I and III collagen in rat liver tissues induced by CCl4, and exert significant anti-hepatic fibrosis. This effect is mainly attributed to the fact that BMSC inhibits HSC activation and induces activated HSC apoptosis. It was observed in the [26], a TGFβ1-induced human HSC activation cell model [26], after BMSC intervention, the activation ability of HSC was significantly weakened, and the expression of α-SMA and type I collagen was significantly reduced. BMSC can act against hepatic fibrosis by regulating multiple signaling pathways on HSC. After transplanting BMSCs in rats with cirrhosis induced by thioacetamide , the expression levels of TGFβ1 and its downstream receptor Smad3 in liver tissue were significantly reduced, and the expression of α-SMA was reduced, suggesting that BMSC can inhibit HSC activation by regulating the TGFβ/Smad signaling pathway and thus improve liver fibrosis [27]. NADPH oxidase is the main source of free radicals (ROS), and ROS is involved in the pathological process of liver fibrosis. BMSC reduces the expression of p47phox, a subtype of ROS marker 4-HNE and NOX in HSC, and inhibits HSC activation of [26] by regulating the NADPH oxidase pathway. In addition, the synthesis and degradation of extracellular matrix is regulated by matrix metalloproteinase (MMP) and metalloproteinase tissue inhibitor (TIMP). BMSC transplantation can upregulate the expression of MMP-9 in liver tissue, downregulate the expression of TIMP-1 in liver tissue, and promote extracellular matrix degradation [28].
BMSC-Ex can also effectively interfere with the HSC activation process and prevent the progress of liver fibrosis. In the rat model of liver fibrosis induced by CCl4 [29], human-derived BMSC-Ex can inhibit Wnt/β-catenin pathway signal transduction in HSCs, reduce HSC proliferation and activation, and thereby reduce liver fibrosis. In addition, exosomes can be delivered between cells as signal delivery vehicles. The circular DNA circDIDO1 was transfected with plasmid and then circDIDO1 was successfully packaged into BMSC-Ex. The BMSC-Ex carrying circDIDO1 was isolated and co-incubated with LX2 cells. It was found that BMSC-Ex-mediated circDIDO1 transfer was upregulated by PTEN to inhibit the activity of AKT signaling pathway, thereby inhibiting the activation of HSC [30].
3.3 BMSC participates in immune regulation
BMSC has strong ability to regulate immune . By improving the status of liver immune cells, it inhibits the secretion of proinflammatory factors and reduces inflammatory damage in the liver. macrophages are the main players in innate immunity, and M1 macrophages are generally believed to have proinflammatory effects, while M2 macrophages have anti-inflammatory effects. In the mouse model of liver fibrosis induced by CCl4, [31], BMSC intervention promoted macrophages to polarize to M2 subtype and inhibited their polarization to M1 subtype, thereby reducing the expression of inflammatory factors IL-12b, IFNγ, TNFα and IL-6, and alleviating the inflammatory response. Similarly, after BMSC intervened in Con-A-induced liver injury mice, the number of M2 macrophages increased, and serum IL-10 levels were upregulated and stimulated signaling and transcriptional activation protein 3 phosphorylated , thereby reducing the number of TUNEL-positive cells and protein expression of cleaved-Caspase3/8/9 in liver tissue, inhibiting hepatocyte apoptosis [32]. BMSC can also regulate the Hedgehog/SMO/Gli1 pathway and Notch1 signaling to inhibit inflammation. The direct binding of CD47 on BMSC and SIRPα in macrophages can induce nuclear translocation of Gli1 and Notch1 intracellular domains (NICDs) in macrophages, while NICD interacts with Gli1 and regulates its target gene Dvl2, thereby inhibiting the inflammatory response driven by NLRP3 inflammasomes [33]. After T lymphocytes are activated, they proliferate and differentiate in large quantities, and at the same time release proinflammatory factors to exert their effector functions [34].BMSC inhibits the differentiation of liver T lymphocytes into the CD4+ subtype, thereby reducing the expression of serum proinflammatory factors and α-SMA in liver tissues, and alleviating inflammation and fibrosis [35]. Similarly, BMSCs transfected with the lentiviral vector carrying Becn1-shRNA also showed the ability to inhibit the proliferation of CD4+ and CD8+T lymphocytes [36]. In addition, implanting the liver decellularized biological scaffold into rats, peripheral infusion of BMSC can reduce neutrophil infiltration and fibrous tissue formation, but this effect can only last for about 2 weeks [37].
In the environment of cholestatic liver injury, the expression of liver vascular endothelial adhesion molecule 1 is increased, recruiting neutrophils and CD8+T lymphocytes, promoting inflammatory damage and fibrosis around biliary duct ; while BMSC-Ex can reduce the expression of vascular endothelial adhesion molecule 1, reduce immune cell infiltration, and protect the liver from inflammatory damage [38]. In addition, miRNA contained in exosomes is one of the important biologically active substances that it plays a role. [39] was studied and found that transfecting BMSC-Ex overexpressing miR-223 using lentivirus can improve liver function and liver tissue inflammatory damage in mice, and significantly reduce the expression of NLRP3 and Caspase-1 in liver tissue. However, after inhibiting the expression of miR-223 in BMSC-Ex, its therapeutic effect disappeared, suggesting that miR-223 in BMSC-Ex plays an anti-inflammatory role by inhibiting the NLRP3 inflammasome signaling pathway.
4 The role of traditional Chinese medicine combined with BMSC transplantation in ESLD
In recent years, a large number of studies have focused on how to improve the clinical effect of BMSC in treating ESLD. Common methods such as gene editing technology, cytokines or growth factor pretreatment of BMSC, but gene reprogramming has potential safety risks. It is worth noting that there have been research on [41-42] that shows that traditional Chinese medicine can promote the proliferation and differentiation ability of BMSC, and has the advantages of low cost and high safety. It is expected to develop an auxiliary treatment application for potential BMSC transplantation.
More and more studies have shown that the combination of traditional Chinese medicine compound prescriptions and BMSC in ESLD has a synergistic effect. For example, BMSC combined with Yiguan Dan [43], Biejia Dan Wan [44], and Jisheng Shenqi Wan [45] can all improve the liver function of rats with CCl4-induced cirrhosis and reduce liver fibrosis. The effect of the combination of the two is better than BMSC transplantation alone. Further research has found that the Chinese medicine compound and its active ingredients can enhance the ability of BMSC to the damaged liver and differentiate into hepatocytes. For example, in the rat model of cirrhosis induced by CCl4 [41], the number of green fluorescent protein-labeled BMSC in liver tissue in the BMSC transplant group combined with CCl4 was significantly higher than that in the BMSC transplant group alone. Rat BMSCs were cultured with cordyceps polysaccharide drug-containing serum in vitro. It was found that AFP expression was visible in the cells of the Cordyceps polysaccharide group at 7 days, and cells at 14 days expressed CK18 and Alb, suggesting that Cordyceps polysaccharide can induce BMSCs to differentiate into hepatocytes [42].
research group used bile duct ligation to induce cholestatic liver fibrosis model in rats in the early stage. It was found that the activation of the Notch signaling pathway in the liver and promote the differentiation of liver progenitor cells into bile duct epithelial cells. The expression level of the negative regulator of the Notch signaling pathway decreased, blocking Notch signaling can inhibit the progression of liver fibrosis [46]. The intervention of Huangqi Decoction can significantly inhibit the activation of Notch signal and the progress of liver fibrosis, and significantly increase the expression level of Numb [47]. BMSC and Numb knockdown BMSC (BMSCNumb-KD) were further injected into rat liver through the spleen while bile duct ligation. It was found that BMSC transplantation can significantly reduce collagen deposition in liver tissues and inhibit bile duct response and HSC activation, suggesting that BMSC transplantation can inhibit the progression of bile liver fibrosis; and after BMSCNumb-KD transplantation, it can be seen that it mainly differentiates into bile duct epithelial cells, promoting bile duct response and liver fibrosis progression, suggesting that Numb plays an important role in the occurrence and development of bile liver fibrosis. Moreover, when BMSCNumb-KD cells were transplanted, Huangqi Decoction was given intervention. Although the degree of liver fibrosis improved, the Hyp content, the positive area of Sirius red collagen staining, the expression levels of α-SMA, CK7 and CK19 were significantly higher than those of BMSC and Huangqi Decoction alone, suggesting that Numb may be a potential target of Huangqi Decoction for anti-biratory liver fibrosis [48].
5 Problem and Prospect
BMSC and its exosomes have unique advantages in the treatment of ESLD, especially most clinical trials have shown the safety and effectiveness of BMSC transplantation. However, there are still some studies that chromosomal aberrations may occur when culturing BMSCs in vitro, and long-term in vitro culture will reduce the proliferation and differentiation ability of BMSCs [50]. It is still necessary to conduct randomized controlled clinical trials with large samples to improve the quality of clinical research evidence. The treatment of ESLD for BMSC-Ex is currently in the basic research stage, and the qualitative and precise biological mechanisms of exosomes still need to be further analyzed. It is worth noting that autologous BMSC is mobilized from the bone marrow during liver injury and migrated to the damaged liver through peripheral circulation. It is a prerequisite for its function. How to mobilize autologous BMSC to the liver and promote liver tissue repair and regeneration can avoid the disadvantages brought by BMSC transplantation and is an important idea for the treatment of ESLD. The compound prescriptions of traditional Chinese medicine and their active ingredients can improve the liver microenvironment, which is crucial for the ability of BMSC to homage to the liver and differentiate. Therefore, in-depth exploration of the traditional Chinese medicine compound and its effective ingredients, and how to mobilize autologous BMSC to the liver and repair damaged liver tissue to relieve fibrosis are extremely challenging and meaningful research directions in the future.
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http://www.lcgdbzz.org/cn/article/doi/10.3969/j.issn.1001-5256.2022.11.041
Citation This article Citation
Liang Yue, Hu Yonghong, Liu Wei, et al. Research progress in the treatment of end-stage liver disease by bone marrow mesenchymal stem cells and its exosomes [J]. Journal of Clinical Hepatobiliary Disease , 2022, 38(11): 2643-2648.
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Special attention | Molecular mechanism of liver injury in acute pancreatitis
Special attention | Factors influencing serological conversion of HBeAg in patients with chronic hepatitis B Special attention | Effects of antiviral treatment on reversal of liver fibrosis
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