Recent studies have shown that ADSCs transplantation can alleviate hepatic I/R combined with hepatectomy injury by reducing the level of hepatocyte autophagy. In addition, in hepatic I/R combined with hepatectomy injury, undisciplined autophagy leads to the accumulation of autophagy-related proteins, which leads to the death of hepatocytes[11, 12]. Consistent with previous studies, we observed that ADSC-CM improved the pathological and microscopic changes of liver parenchyma in porcine hepatic I/R combined with hepatectomy model by alleviating hepatocyte autophagy.
Autophagy is an intracellular degradation system that transports components of the cytoplasm into lysosomes for degradation. Autophagy is also a highly conserved biological process that maintains cell homeostasis and viability by recycling and reusing energy. The liver is largely dependent on pathological or physiological autophagy. However, in extreme cases, such as acute organ injury or reperfusion injury[11, 12], undisciplined autophagy can lead to the accumulation of autophagic vacuoles, which can lead to cell death.
Beclin-1, one of the first mammalian autophagy effectors, is involved in mammalian autophagy Beclin-1 recruits PI3KC3 (Vps34) to form a protein complex[30, 31], which in turn regulates intracellular transport and autophagosome formation. The formation of Beclin-1 complexes opens the production of autophagosome membranes. Beclin-1 also regulates autophagic activity[32, 33]. The execution of autophagy requires two ubiquitin-like conjugation systems: the ATG8 (LC3) conjugation system and the ATG12-ATG5 conjugation system. LC3 is the only mammalian protein known to bind stably to the autophagosome membrane. After the synthesis of LC3 protein, its carboxyl end is immediately sheared by ATG4 to produce cytosolically localized LC3I. In the process of autophagy, LC3I is modified by ubiquitination processing and binds to PE on the autophagosome membrane to form membrane-bound LC3II, which is localized in the autophagosome.LC3II present in autophagosomes is one of the molecular markers of autophagy, and the content of LC3II is proportional to the degree of autophagy.ATG7 activates ATG12 and binds to ATG5 under the action of ATG10 to form ATG12-ATG5 complex. In ubiquitin-binding reactions, the ATG5-ATG12 complex promotes the formation of ATG8-PE in a manner similar to the function of E3 enzymes. The formation of ATG8-PE and ATG16-ATG 5-ATG12 complexes is necessary for the formation of autophagosomes. The multifunctional protein p62 (also known as SQSTM1) plays an important role in signaling and selective autophagy. Therefore, we examined the gene and protein expression of autophagy-related factors Beclin-1, LC3Ⅱ, ATG5, ATG12, p62 to observe the changes of autophagy activity induced by hepatic I/R combined with hepatectomy. We observed an increase in the Beclin-1, ATG5, ATG12 mRNA and the Beclin-1, LC3Ⅱ protein levels, and an decrease in the p62 mRNA and protein levels in the liver tissues after IRI, which were significantly alleviated by the ADSC-CM. This clearly indicated that autophagy overexpression was induced in the hepatic I/R combined with hepatectomy and alleviated by injecting ADSC-CM.
Autophagy plays an important role in hepatic IRI[11, 12], and the mechanism target of PI3K/Akt/mTOR signaling pathway can regulate cell autophagy[35, 36]. PI3K-AKT controls cellular functions, including mTOR, by regulating the expression of many downstream molecules. mTOR is a molecular target of rapamycin in mammalian cells and an important factor regulating cell growth and metabolism. Activation of mTOR can inhibit the expression of autophagy. Phosphorylated AKT activates mTOR, which negatively regulates autophagy by inhibiting the downstream molecule ULK1 complex. Here, hepatic I/R combined with hepatectomy injury inhibited the PI3K/Akt/mTOR pathway. ADSC-CM treatment activated the PI3K/Akt/mTOR pathway, upregulated the gene expression of PI3K, AKT and mTOR and the protein expression of p-AKT and p-mTOR in the liver. Therefore, combined with the above autophagy-related results, it is concluded that ADSC-CM may inhibit the excessive autophagy of hepatocytes after hepatic I/R combined with partial hepatectomy by regulating the PI3K/Akt/mTOR pathway to alleviate the autophagy injury of hepatocytes.
ADSC-CM is the supernatant concentrate of ADSCs collected at rest in serum-free culture medium, which contains a variety of growth factors, chemokines, cytokines and extracellular vesicles secreted by ADSCs in paracrine or autocrine ways[22, 38]. ADSCs secretome can stimulate cell proliferation, inhibit apoptosis, promote angiogenesis, inhibit inflammation and immune response. And it not only overcomes the limitations of cell therapy, but also maintains its advantages. Therefore, these cell-free products have the potential to replace cell transplantation therapies. The results of comparison of ADSC-CM and ADSCs in the regulation of hepatic IRI-induced excessive autophagy showed that after transplantation of ADSC-CM and ADSCs, the expression of excessive autophagy in hepatocytes decreased, and there was no significant difference between them. Therefore, both ADSC-CM and ADSCs have protective effects on hepatic autophagy injury, and ADSCs may regulate and treat autophagy injury caused by hepatic I/R combined with hepatectomy in miniature pigs through paracrine effect.