*2.2. Isorhamnetin Treatment Protects Mice from E. coli-Induced Inflammation*

The effect of isorhamnetin on *E. coli*-induced myeloperoxidase (MPO) and proinflammatory cytokines (TNF-α and IL-6) was then examined. As depicted in Figure 3A,B, MPO activity is high in untreated mice infected with *E. coli*, whereas treatment with isorhamnetin decreased MPO activity in the kidney, liver and lung. However, the best results were obtained in the lung tissue (Figure 3B). Moreover, the secretion of cytokines is also highly increased in the sera and lung lysates after *E. coli* infection, which was drastically reduced in the isorhamnetin-treated group, suggesting that isorhamnetin can suppress the inflammatory response (Figure 3C,D).

**Figure 3.** In vivo anti-inflammatory and toxicity evaluation of isorhamnetin. (**A**,**B**) Examination of myeloperoxidase (MPO) activity in lung, liver and kidney. (**C**,**D**) Production of cytokines (TNF-α, IL-6) in the sera and lung lysates. (**E**) Aspartate aminotransferase (AST), alanine amino transferase (ALT) and blood urea nitrogen (BUN) levels in the serum. (F) Hematoxylin and eosin staining of the lung tissue. Data are presented as the means ± SEM. \* *p* < 0.05; \*\* *p* < 0.01; and \*\*\* *p* < 0.001 compared to the *E. coli* group.

Additionally, aspartate aminotransferase (AST), alanine amino transferase (ALT) and blood urea nitrogen (BUN) assays were carried out to evaluate liver and kidney function, and the results of these assays reveal that untreated mice infected with *E. coli* develop severe liver and kidney dysfunction (Figure 3E). However, isorhamnetin treatment protects the mouse liver and kidneys from *E. coli*-induced inflammation, as indicated by the reduced levels of AST, ALT and BUN (Figure 3E). Notably, we also examined the effect of isorhamnetin on uninfected mice to assess toxicity; here, we observed that this treatment does not alter the levels of AST, ALT and BUN, compared to those in the control group (Figure 3E), clearly indicating its safety. Moreover, we examined the histopathology of the lung tissues to assess neutrophil infiltration; the results indicate that isorhamnetin treatment could reduce the infiltration of neutrophils in the lung, which is noticeable after *E. coli* treatment (Figure 3F).
