Prolonged inflammation is a common hallmark of fibrosis, represented by the release of a wide range of growth factors and inflammatory mediators, such as TGF-β, IL-17, and IL-13, to stimulate organ fibrosis, including LF . However, TGF-β is the most well-known mediators that initiate the complex pathways involved in LF . The prolonged expression of TGF-β consistently results in the activation and differentiation of HSCs into active MFs, characterized by α-SMA expression, and resulting in LF formation . Furthermore, our previous study demonstrated that MSCs can migrate to liver injury sites , to regenerate liver damage and restore liver structure and function following LF . In the same time, MSCs can suppress inflammation by releasing anti-inflammatory cytokines, particularly IL-10 . However, the mechanism through which MSCs improve and restore LF by controlling TGF-β and α-SMA expression remains unclear. Therefore, this study investigated the role played by MSCs in the amelioration of LF by suppressing the release of TGF-β release, leading to decreased α-SMA expression, 3 and 14 days after treatment.
We used CCl4 as a hepatotoxic chemical to induce LF in an experimental animal model because the CCl4 model represents the model that most closely resembles human liver cirrhosis . After 8 weeks of CCl4 injections, we successfully established a rat model of LF . To confirm the LF in this study we used Sirius Red staining to identify collagen fibers, and we found a significant increase in the percentage of collagen fibers in several areas, indicating the successful induction of LF in all study groups (Fig. 4a, b). LF is characterized by chronic inflammation and the massive recruitment of Th2 lymphocytes, which induce the polarization of macrophage type 1 (M1) into macrophage type 2 (M2) cells, through the release of IL-13 and IL-4 . Active M2 and Th2 cells release TGF-β to activate quiescent HSCs (inactivate HSCs), which differentiate into MFs (active HSC) and produce large amounts of ECM . Therefore, to analyze the role played by MSCs in the inactivation of HSCs, we explored the levels of TGF-β following MSCs administration.
Here, we present evidence that MSCs can suppress the release of TGF-β in an LF model animal. We believe this discovery is both novel and important discovery because it demonstrates mechanistically how fully developed TGF-β-dependent fibrosis can be disrupted by MSCs treatment. In this study, using a CCl4-induced LF animal model, we showed that MSCs can decrease TGF-β levels, released by Kupffer cells and M2 macrophages. We suggest that MSCs regulate TGF-β release through immunomodulatory mechanisms, by releasing several anti-inflammatory cytokines, particularly IL-10. The binding of IL-10 with receptors on Kupffer cells and M2 macrophages might activate tyrosine kinase 2 and Janus tyrosine kinase 1 (JAK1), which phosphorylate IL-10Rα and signal transducer and activator of transcription 3 (STAT3). The phosphorylated STAT3 translocates into the nucleus, where it binds the promoters of various IL-10 target genes, particularly the suppressor of cytokine signaling 3 (SOCS3) whose expression has been correlated with the decreased expression of tumor necrosis factor (TNF)-α, IL-1β, and TGF-β  (Fig. 2). These findings were confirmed by our previous study, which reported that MSCs can prevent peritoneal fibrosis by releasing IL-10 . Furthermore, other studies have also reported that IL-10 plays a crucial role in the inhibition of fibrosis-related inflammation . TNF-α-exposed MSCs can release IL-10 to control inflammatory cytokine release . To analyze the effects of decreased levels of TGF-β following MSCs treatment on HSCs activity, we assessed α-SMA expression.
LF was evidenced by the significant increase in the percentage area percentage of collagen fibers produced by active HSCs, which represent the primary cell type responsible for fibrogenesis. Activated HSCs are characterized by α-SMA expression . The prolonged release of TGF-β associated with CCl4-induced hepatocyte damage can activate quiescent HSCs to differentiate into the star-shaped stellate cells or into MF-like cells, which synthesize large quantities of ECM components, including collagen, proteoglycan, and adhesive glycoproteins . Our findings revealed that LF was also resolved after MSCs administration, associated with a significant decrease in the area of α-SMA-positive cells, which significantly decreased compared with the control group, similar to the observed decrease in TGF-β levels (Fig. 4c, d). We suggest that MSCs release IL-10, to downregulate profibrotic genes and upregulate anti-fibrotic hepatic genes . Moreover, IL-10-released by MSCs may act as a receptor-binding competitor for TGF-β in quiescent HSCs; thus, IL-10 may play an inhibitory role in process HSC transition from the quiescent state to the activated state, in addition to inducing the apoptosis of active HSCs . These findings were confirmed by another study, which reported that MSCs can reduce the expression levels of collagen type I and α-SMA .
In summary, the administration of MSCs attenuated LF 14 days after MSCs administration by reducing TGF-β concentrations and α-SMA expression. However, whether IL-10 was the primary actor associated with the reductions in TGF-β concentration and α-SMA expression) was not measured and represent a limitation of this study.