Mechanism of Huoxue Rongluo Formula Mediated Bmal1 Regulation of Necroptosis After Ischemic Stroke

OBJECTIVE  To investigate the mechanism on Huoxue Rongluo formula mediated Bmal1 regulation of necroptosis after ischemic stroke.

METHODS  Male C57BL/6 mice were randomly assigned to the sham operation group, ischemic stroke model group, circadian disruption composite model group, edaravone group, and low-, medium-, high-dose Huoxue Rongluo formula groups. Among them, in the sham operation group, only the blood vessels were separated without inserting the suture. In the ischemic stroke model group, only the preparation of middle cerebral artery occlusion(MCAO) was performed. The environmental circadian disruption(ECD) and MCAO model were respectively established in the circadian disruption composite model group, the edaravone group, and the low-, medium-, high-dose Huoxue Rongluo formula groups. Modified neurological severity score(mNSS) score and 2,3,5-triphenyltetrazolium chloride staining were used to evaluate the effects of ECD and Huoxue Rongluo formula on neurological deficit symptoms and cerebral infarct volume in each group of mice. HE staining and Nissl staining were used to evaluate the pathological damage in the hippocampus of mice in each group. ELISA was used to detect the levels of TNF-α, IL-1β and IL-6. Immunofluorescence was used to detect the expression levels of Bmal1, p-RIPK3 and p-MLKL in the hippocampus of mice in each group. Western blotting was used to detect the differences in protein expression of Bmal1, RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL and p-MLKL among the groups.

RESULTS Comapred with ischemic stroke model group, the mNSS and infarct volume in the circadian disruption composite model group were significantly increased(P<0.01), the structural damage, disordered arrangement and vacuolar necrosis of neurons and glial cells in the hippocampus were significantly increased, the number of Nissl bodies was significantly decreased(P<0.05), the level of inflammatory factors was significantly increased(P<0.01 or P<0.05), and the expression of related proteins in brain tissues was significantly different from that in ischemic stroke model group(P<0.001, P<0.01 or P<0.05). The mNSS, infarction volume, arrangement of neurons and glial cells in the hippocampal region, and the number of Nissl bodies in each dose group of the Huoxue Rongluo formula were all improved to varying degrees compared with the circadian disruption composite model group. Among them, the high-dose group of Huoxue Rongluo formula showed the most significant improvement(P<0.01 or P<0.001), and there were significant differences in the levels of inflammatory factors and the expression of related proteins(P<0.001).

CONCLUSION ECD can intensify the process of necrotizing apoptosis after ischemic stroke, and Huoxue Rongluo formula can regulate necrotizing apoptosis after ischemic stroke by targeting Bmal1 to reduce the inflammatory cascade after ischemic stroke.