*3.5. Blockade of CB1 Significantly Attenuates Neutrophil Infiltration and Liver Inflammation in CCl4-Treated Mice*

The effects of CB1 blockade on neutrophil function and liver inflammation were further verified in vivo in mice treated with CCl4 for 2 weeks, when the hepatic levels of neutrophil signatures were highest. CB1 blockade by the administration of AM281 restrained the mRNA levels of Ly6G in injured livers (Figure 6A). In line with this, FACS analysis showed decreased neutrophils infiltration after AM281 administration in CCl4-treated mice compared with CCl4-treated alone (Figure 6B,C). Similarly, the down-regulated expression of CitH3 (Figure 6D,E) was observed in CCl4-treated mice with the administration of AM281, indicating less formation of NETs. Representative H&E-stained images showed a significant decrease of infiltrated inflammatory cells (Figure 6F) and the inflammatory area was quantified by digital image analysis (Figure 6G). Besides, AM281 administration protected liver against CCl4-induced injury with lower levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum (Figure 6H). To elucidate the significance of NETs formation in liver inflamamtion, DNase I was administrated in CCl4-treated mice. Our results showed that DNase I significantly reduced mRNA expression of markers for liver inflammation (MCP1, IL-1β, IL-6) and macrophage activation (CD86) in the liver of CCl4-treated mice (Figure 6I). Altogether these results demonstrate that blockade of CB1 inhibits the recruitment and activation of neutrophils in the early stage of chronic liver injury and significantly attenuates liver injury.

**Figure 6.** Blockade of CB1 significantly attenuates neutrophil infiltration and liver inflammation in CCl4-treated mice. (**A**) Ly6G mRNA expression in CCl4- or olive oil (OO)-treated liver with or without the administration of AM281. (**B**,**C**) Representative FACS plots and quantification for neutrophils in liver. (**D**,**E**) CitH3 protein levels in liver. (**F**) Representative H&E staining images of liver sections. Scale bars, 100 μm. (**G**) Quantitative analysis of liver inflammation areas. (**H**) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by chemistry analyzer. (**I**) mRNA expression of MCP-1, IL-1β, IL-6, and CD86 in CCl4- or OO-treated liver with or without the administration of DNase I. Data are presented as the mean ± SEM. N = 6 per group. \* *p* < 0.05 vs. OO. # *p* < 0.05 vs. CCl4-treated alone.
