Abstract:

Objective To investigate the hepatoprotective effect and anti-hepatocyte pyroptosis effect of Yinchenhao decoction synergized with umbilical cord mesenchymal stem cells （UcMSCs） in vitro and vivo models of acute liver failure （ALF）， as compared to single?drug treatment. Methods UcMSCs were extracted from umbilical cords and identified using electron microscopy and flow cytometry. Then the exosomes released from ucMSCs （UcMSCs?exo） were isolated through ultracentrifugation， and ucMSCs?exo were identified by nanoparticle tracking analysis （NTA）， transmission electron microscopy， and Western blot for CD63 and CD9. Human hepatocyte cell line LO₂ cells were stimulated by 100 ng/mL lipopolysaccharide （LPS） and 5 mmol/L adenosine triphosphate （ATP） to construct a vitro model of ALF. The cells were randomly divided into the following groups： a normal control group， a model group， a Yinchenhao decoction treatment group， a ucMSCs?exo treatment group， and a combination treatment group. The morphological changes of LO₂ were observed under the electron microscope， the mortality of LO₂ cells was detected by flow analysis， the expression of pyroptosis?related proteins Caspase?1 and Cleaved?Caspase?1 was detected by Western blot， and the expression of pyroptosis?related cytokines IL?1β and IL?18 was detected by ELISA. Then， fifty 6?8?week?old male C57BL/6 mice were randomly divided into the following groups： normal control group， model group， Yinchenhao decoction treatment group， ucMSCs?exo treatment group， and combined treatment group. Each group consisted of 10 mice. The ALF mouse model was constructed by intraperitoneal injection of 10 μg/kg LPS and 800 mg/kg D?galactosamine （D?GalN）. After successful modeling， each group was given the appropriate drug interventions， and the ucMSCs?exo and combined treatment groups were injected with ucMSCs?exo interventions in the tail vein， and the drugs were administered continuously for 3 days. After 12 hours of intervention， serum specimens and liver tissues were collected from each group. The expression levels of ALT and AST were detected， and the liver tissues were stained with HE to observe the pathological changes. Results In the ALF cell model， hepatic cell death was inhibited in the Yinchenhao decoction treatment group， the ucMSCs?exo treatment group， and the combination group （P < 0.05）. The rate of hepatic cell death in the combination treatment group was significantly reduced （P < 0.05）. Initially， the study aimed to explore the effect on hepatic cell pyroptosis， and it was found that the combination treatment group could effectively suppress hepatocellular cell death more effectively compared to the single treatment groups. The combined treatment group significantly reduced the expression of the pyroptosis?related protein Cleaved?Caspase?1， as well as the cytokines IL?1β and IL?18 （P < 0.05）. However， there was no statistically significant difference in the expression of Caspase?1 （P > 0.05）. In the vivo model， the single Yinchenhao decoction treatment group， the ucMSCs?exo treatment group， and the combined treatment group were able to effectively reduce the serum ALT and AST levels compared to the model group（P < 0.05）. Additionally， these treatments were found to attenuate hepatocellular necrosis and the infiltration of inflammatory cells. Compared to the group treated with only Yinchenhao decoction or only ucMSCs?Exo， the group that received combined treatment showed a greater decrease in serum ALT and AST levels. Additionally， there was a reduction in the infiltration of inflammatory cells and the extent of cell death in the liver tissues （P < 0.05）. Conclusions The combination of Yinchenhao decoction and ucMSCs?exo demonstrated a significantly better hepatoprotective effect in the ALF vivo model compared to the individual treatments of Yinchenhao decoction or ucMSCs?exo alone. Furthermore， this combination treatment was able to effectively inhibit hepatocyte pyroptosis.

Key words: acute liver failure, Yinchenhao decoction, umbilical cord mesenchymal stem cells, hepatocyte pyroptosis, exosomes