Mechanism of action of Zhuyu Annao pill in mice with cerebral intrahemorrhage based on TLR4.

OBJECTIVE: To explore the protective effect and possible mechanism of action of Zhuyu Annao pill in mice with intracerebral hemorrhage (ICH).

METHODS: Sixty mice were divided into the control group, hemorrhage group, drug-treated group (after hemorrhage), TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group (after hemorrhage) with 12 in each group. Model of autologous ICH was established in all groups. After drilling and 12 h of fasting, models in the control group hemorrhage group and TLR4-knockout hemorrhage group were all drenched with 10 mL/kg distilled water by intragastric administration. Models in the drug-treated group and TLR4-knockout hemorrhage + drug-treated group were drenched with 6.25 g/kg of Zhuyu Annao pill. All groups were treated for 7 d. Longa scoring method was used to measure the neurological defect scores and determine the brain water contents of all groups; ELISA was employed to detect the inflammatory factor interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and IL-1β in brain tissues; and Western blot was applied to test the expression quantities of apoptotic protein Bax and anti-apoptotic protein Bcl-2 in brain tissues.

RESULTS: At day 3 and 7, compared with the hemorrhage group, the neurological defect scores of the drug-treated group, TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group decreased significantly (P < 0.05). Compared with the hemorrhage group, the brain water contents of the drug-treated group, TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group reduced significantly (P < 0.05). Compared with the hemorrhage group, the inflammatory factor IL-6, TNF-α and IL-1β of the drug-treated group, TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group decreased significantly (P < 0.05). Compared with the hemorrhage group, the expression of apoptotic protein Bax of the drug-treated group, TLR4-knockout hemorrhage group and TLR4-knockout hemorrhage + drug-treated group decreased significantly and the expression of anti-apoptotic protein Bcl-2 increased significantly (P < 0.05).

CONCLUSIONS: Zhuyu Annao pill can alleviate encephaledema for mice with ICH and reduce inflammatory responses and nerve cell apoptosis. TLR4 can mediate inflammatory injury induced by ICH. Thus, Zhuyu Annao pill can play a protective role for brains by decreasing the expression of TLR4.