The Mechanism of Huoxue Rongluo Formula Combined with Edaravone and Dexborneol in Regulating Mitochondrial Autophagy-NLRP3 Inflammasome to Alleviate Damage in HCMEC/D3 Cells

SU Qihou; LI Zhong; ZHOU Desheng; ZHANG Yuxing; TANG Ning; ZENG Fukang

doi:10.12102/j.issn.1672-1349.2026.02.006

Abstract：Objective:To explore the mechanism of Huoxue Rongluo Formula combined with edaravone and dexborneol in interfering with the mitochondrial autophagy-NOD-like receptor family Pyrin domain containing 3(NLRP3)inflammasome pathway in human brain microvascular endothelial cells(HCMEC/D3)with the oxygen-glucose deprivation/reoxygenation(OGD/R)model.Methods:Through in vitro experiments,HCMEC/D3 was divided into the normal group,the model group,the Huoxue Rongluo Formula group,the edaravone and dexborneol group and the combination medication group.By reviewing literatures,the optimal modeling time was determined.Cell viability was detected using the cell counting kit(CCK8),and the optimal drug concentration and combination were identified.After clarifying the optimal modeling time,the drug concentration and drug combination,in vitro experiments were conducted to investigate the effects on the expression of PTEN-induced kinase 1(PINK1)/Parkin in the autophagy pathway and NLRP3/Caspase-1 in the inflammatory pathway.The effect of inflammatory factors interleukin(IL)-1β and IL-18 in the cell supernatant was detected by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR) was used to detect the effect of drugs on the expression of PINK1/Parkin mRNA in the autophagy pathway.Western Blot was used to detect the effect of the drug on the expression of PINK1/Parkin mRNA and NLRP3/Caspase-1 in the autophagy pathway. Results:The original solution of edaravone and dexborneol injection could inhibit the activity of HCMEC/D3(P<0.05 or P<0.01).The optimal therapeutic effect of edaravone and dexborneol was achieved at a concentration of 20 mL/L,confirming 20 mL/L was the optimal drug concentration.The results of the CCK8 method showed that compared with the model group,the HCMEC/D3 cell viability in the Huoxue Rongluo Formula group,the edaravone and dexborneol group,and the combined medication group increased(P<0.01).Compared with the Huoxue Rongluo Formula group,the combined medication group promoted an increase in the viability of HCMEC/D3 cells(P<0.01).Compared with the edaravone and dexborneol group,the HCMEC/D3 cell viability in the combined medication group increased(P<0.01).It was confirmed that the combination medication group was the best drug combination for the experimental study.Compared with the normal group,the expressions of inflammatory factors IL-1β,IL-18,and PINK1/Parkin mRNA,the protein expressions of inflammatory pathway NLRP3/Caspase-1,and the protein expressions of autophagy pathway PINK1/Parkin in the model group increased(P<0.05 or P<0.01).Compared with the model group,the levels of inflammatory factors IL-1β and IL-18 in the combined medication group decreased,while the levels of inflammatory factors in the autophagy inhibition group increased.The expressions of PINK1/Parkin mRNA in the combination medication group increased(P<0.05),while those in the autophagy inhibitor group decreased(P<0.01).The protein expression of inflammatory pathway NLRP3/Caspase-1 in the combination medication group decreased(P<0.05),while the protein expression of autophagy pathway PINK1/Parkin increased(P<0.05 or P<0.01),and the protein expression of inflammatory pathway NLRP3/Caspase-1 in the autophagy inhibitor group increased(P<0.05).The protein expression of PINK1/Parkin in the autophagy pathway decreased(P<0.05). Conclusion:Edaravone and dexborneol combined with Huoxue Rongluo Formula might play neuroprotective effects by promoting mitochondrial autophagy to regulate NLRP3 inflammasome activation.