Title: Baicalin regulates autophagy to interfere with small intestinal acute graft-versus-host disease

Abstract

Acute graft-versus-host disease (aGVHD) is the main complication and the cause of death after allogeneic hematopoietic stem cell transplantation. Previous studies have shown that interference with autophagy may against aGVHD after bone marrow transplantation. Baicalin can protect the small intestinal epithelial cells of rats against TNF-α-induced injury and alleviate enteritis-related diarrhea. To verify whether baicalin can protect the small intestinal mucosal barrier by regulating abnormal autophagy, a mouse model of intestinal aGVHD was established. CB6F1 mice were intravenously injected with a suspension of mononuclear cells derived from BALB/c donor mice bone marrow and splenic tissue after treatment with 60Co X-rays. The survival time, TNF-α and IL-10 levels, and autophagy markers levels in the intestine were assessed after treatment with different doses of baicalin for 15 days. A cell model of intestinal barrier dysfunction was also constructed to verify the effect of baicalin in vitro. The results showed that baicalin significantly reduced the aGVHD pathology score and clinical score, and prolonged the survival time of aGVHD mice by decreasing the TNF-α level and increasing the IL10 level. Immunofluorescence staining, immunohistochemistry, western blot, and transmission electron microscopy examination showed that baicalin treatment increased the autophagy level and led to the recovery of mitochondrial structures in the intestinal mucosal epithelial cells of mice and Caco-2 cells. Western blotting results also showed that baicalin enhanced autophagy by regulating the AMPK/mTOR pathway, which were observed both in vivo and in vitro. Furthermore, the effect of baicalin was reduced after combination treatment with the autophagy inhibitor 3-methyladenine. In summary, baicalin can alleviate the severity of small intestinal aGVHD and intestinal mucosal barrier damage by regulating autophagy and inflammatory cytokine levels, thus may have the potential to be a new treatment for aGVHD.