[Menstrual blood-derived mesenchymal stem cell transplantation improves anti-fibrosis mechanisms in mouse models of alcoholic liver fibrosis].

Objective: To investigate the ameliorative effect of menstrual blood-derived mesenchymal stem cells (MenSCs) on liver fibrosis and injury, and further study whether it plays a role by regulating the Toll-like receptor (TLR) signaling pathway in a mouse model of alcoholic liver disease (ALD). Methods: A C57BL/6J mouse model of ALD was established using the gradient ethanol gavage method combined with pyrazole. A blank control group and a model group were established. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) activities, as well as inflammatory factors, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), were measured. The modeling effect was evaluated by combining Masson staining and Sirius red staining in liver tissue with Western blotting to detect the protein expression of α-smooth muscle actin (α-SMA) and collagen type I α1 chain (COL1A1). Simultaneously, qPCR was used to detect the mRNA expression of TLRs and transforming growth factor β-activated kinase 1 (TAK1) to explore the related mechanisms. ALD model mice were divided into a phosphate-buffered saline (PBS) control group and a MenSCs treatment group [2×10⁵ MenSCs (200 μL) injected via tail vein], with intervention once a week sustained for four weeks after successful modeling. Mice batches were sacrificed on the seventh day following each intervention to collect serum and liver tissues. Collagen deposition changes were observed using Sirius red staining. The expression of α-SMA and transforming growth factor-β1 (TGF-β1) proteins was detected by Western blotting. Serum biochemical indicators levels were also measured. The effects of MenSCs on TLRs and TAK1 mRNA expression were assessed using qPCR to comprehensively evaluate the MenSCs intervention effect. The two groups were compared using paired t-tests. Repeated measures analysis of variance (RM-ANOVA) combined with Tukey's multiple comparisons test was used for comparisons among multiple groups. Results: Compared with the blank control group, serum levels of ALT, AST, and GGT, as well as inflammatory factors TNF-α and IL-6, were significantly elevated, while IL-1β levels were significantly decreased in the mouse model of the ALD group. Collagen deposition in liver tissue was markedly increased. The expression of fibrosis markers α-SMA and COL1A1 proteins was significantly upregulated (α-SMA: P<0.001; COL1A1: P<0.001). The mRNA expression of TLR1, TLR2, TLR4, TLR6, TLR7, and TLR8 was significantly upregulated, and the expression of TLR9 and TAK1 was downregulated, while the expression of TLR3 and TLR5 remained unchanged. Liver function levels (ALT and AST) were significantly reduced; liver fibrosis degree was markedly improved; and TGF-β1 and α-SMA protein expression were suppressed (TGF-β1: P<0.001; α-SMA: P=0.026) following MenSC transplantation in mice. MenSC specifically down-regulated the mRNA expression of TLR1, TLR2, TLR4, TLR6, TLR7, and TLR9, with significant inhibition of TLR1, TLR2, and TLR9 expression (P<0.001), while TLR8 and TAK1 expression were not significantly affected at week 4 of intervention (P>0.05). Conclusion: MenSCs transplantation can effectively improve liver function and reduce fibrosis in a mouse model of ALD, and its mechanism may be related to the inhibition of the TLR signaling pathway activation.