Exosomes of mesenchymal stem cells can be used as a remedy factor to reduce fibrogenic genes and treatment of liver Fibrosis disease.
Exosomes of mesenchymal stem cells can be used as a remedy factor to reduce fibrogenic genes and treatment of liver Fibrosis disease.
Elham Shakerian,1,*
1. 1-Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 2-Department of Clinical Biochemistry, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Introduction: Introduction: Liver fibrosis resulting from chronic liver injury is a major cause of morbidity and mortality in the world. Liver fibrosis is the result of the excessive accumulation of extracellular matrix proteins including collagen that occurs in most chronic liver diseases. Activated hepatic stellate cells (HSCs) have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-β1. Following chronic injury, HSCs activate and secret large amounts of collagen. α-SMA, as a protein, is used as a marker to identify activated fibrogenic cells. α-SMA expression is considered a reliable marker of hepatic stellate cell (HSC) activation and a key biomarker for liver fibrosis. Although many therapeutic interventions are effective in experimental models of liver fibrosis, their efficacy and safety in humans are unknown. So, it is important to know which factors can cause and treat liver fibrosis. Free cholesterol in the diet can cause liver fibrosis by accumulating in Hepatic stellate cells (HSCs). MSCs-derived exosomes are known as the new mechanism of cell-to-cell communication, showing that exosomes can be used as a new treatment. At present, there is little information about the mechanism of exosomes derived from Human umbilical cord Wharton’s jelly (WJ-MSCs) in liver fibrosis. In this study, we investigated the effect of exosomes of WJ-MSCs on the expression of TGF-β, α-SMA, and collagen1α genes in cholesterol-induced liver fibrosis in the LX2 cell line (a type of cell derived from HSCs) and also phosphorylation of Smad3 protein level.
Methods: Methods: First, the LX-2 cells were seeded in 6-well plates (1 × 105 cells per well) in DMEM containing 10% FBS at 37 °C with 5% CO2. When cells are approximately 80–90% confluent, the cells must be sub-cultured to ensure proper growth and health of the cells. Second, after starvation for 16 hr, the cells were treated with 75 and 100 μM cholesterol and incubated for 24 hr to induce liver fibrosis. Isolation, culture, and Differentiation assays of WJ-MSCs were done according to protocol. Next, the exosomes of WJ-MSCs (final concentration of 50 μg/ml) were dissolved in a culture medium and added to the cells for 24 hr. Three groups were considered for the experiment: 1- Control group, 2- Cholesterol treatment group, and 3- Cholesterol treatment group + exosome of WJ-MSCs; after 24 hr, cells were washed twice with PBS, then Real-Time PCR analysis was performed. Western Blots also was done to evaluate the signaling pathway of smad3.
Results: Results: In treatment with 75 and 100 μM cholesterol concentrations, the Real-Time PCR analyses showed that the mRNA expression levels of TGF-β, α-SMA, and Collagen1α (liver fibrosis marker genes) were significantly up-regulated compared to the control group. The mRNA expression of TGF-β, α-SMA, and collagen 1α was significantly increased as a result of the development of cholesterol-induced liver fibrosis. We also found that exosomes of WJ-MSCs treatment (50 μg/ml) could significantly decrease the level of TGF-β, collagen1α, and α-SMA gene expression in cholesterol-induced liver fibrosis model. Treatment with exosomes prevented the activation of HSCs by inhibiting the phosphorylation of the Smad3 protein. the P-Smad3 levels were significantly downregulated after 24 hr, in the treatment of exosomes of WJMSCs in cholesterol-induced HSCs. Treatment with exosomes of WJ-MSCs improves cholesterol-induced liver cell damage.
Conclusion: Conclusion: In our study, exosome treatment effectively downregulated the expression of TGF-β, αSMA, and collagen1α and it also reduced the phosphorylation of Smad3 levels in cholesterol-treated LX2. Thus, suppression of TGF-β expression can improve hepatic fibrosis, and its mechanism of action is through the Smad3 signaling pathway. This study suggests the use of exosomes of WJ-MSC for developing effective therapies for hepatic fibrosis. Cholesterol accumulation in HSCs can activate them and increase fibrosis genes expression, leading to the progression of liver fibrosis. The exosomes of WJ-MSCs can inhibit the TGFβ/Smad3 signaling pathway, and prevent further activation of HSCs and progression of liver fibrosis. So, the exosomes of WJ-MSCs can reduce the expression of fibrosis genes. According to our data, the exosome of WJ-MSCs can be introduced as an effective remedy for liver fibrosis.