The Protective Mechanism Of Puerarin On Brain Injury In Rats With Sepsis By Inhibiting NLRP3/Caspase-1/GSDMD Pyroptosis Pathway And Reducing Blood-brain Barrier Damage

Objective: Sepsis-associated encephalopathy(SAE)is one of the most common complications of sepsis.Up to 70% of sepsis patients will eventually develop to SAE.It is a diffuse brain dysfunction caused by host reaction disorder and no evidence of direct infection,structural abnormality or other types of encephalopathy in the central nervous system.The pathophysiology and potential molecular mechanism of sepsis related encephalopathy have not been fully elucidated,but most scholars believe that the etiology of SAE is multifactorial,and several pathological mechanisms participate in and influence each other,promoting the development of SAE to varying degrees.These factors include ischemia or hemorrhage injury,blood brain barrier destruction,brain microcirculation abnormality,brain metabolism abnormality(including oxidative stress,mitochondrial dysfunction,etc.),a large number of inflammatory factors release,neurotransmitter dysfunction,etc.It is of great clinical significance to explore more therapeutic methods and molecular mechanisms.Puerarin is a isoflavone compound isolated from Puerarin,a traditional Chinese medicine.It has certain therapeutic effects on cardiovascular disease,diabetes,neuroprotection of brain damage,and has a variety of pharmacological effects such as anti-infection,anti-oxidation,and anti-apoptosis.Therefore,it is speculated that it may have a protective effect on sepsis-associated encephalopathy.In this study,the protective effect of puerarin on sepsis brain injury and its possible biological mechanism were studied in vivo and in vitro.Methods: The rat model of septic brain injury was constructed by CLP,and divided into Sham group,CLP group,CLP+Pue group and Pue group.The 72-hour survival rate of rats in each group was observed.ELISA to detect the levels of NSE and S100β,HE staining and Nissl staining were used to evaluate the brain damage in each group.The expression of pyroptosis-related factors was detected by Western Blot,q RT-PCR and immunohistochemistry.The HT22 cells were divided into Control group,LPS+ATP group,and LPS+ATP+Pue group.The expression levels of key factors of pyroptosis were detected by CCK8,Western Blot and q RT-PCR.The pyroptosis of HT22 cells was observed by ELISA and Hoechst33342/PI staining.Changes in the permeability of the BBB were assessed by the detection of brain water content and the detection of EB penetration.Results: The 72-hour survival rate and neurobehavioral scores of rats in the CLP group were significantly lower than those in the other groups.Puerarin helps reduce pathological changes in brain tissue.Compared with the Sham group,the expressions of NLRP3,Caspase-1,GSDMD,cleaved-Capase-1,cleaved-GSDMD,ASC,IL-1β,and IL-18 in the CLP group were increased to varying degrees.Compared with the CLP group,its expression level was lower in mice.The results of in vitro experiments further confirmed that the pyroptosis of HT22 cells treated with puerarin was reduced to varying degrees compared with the pyroptosis model group.The results of BBB-related experiments show that puerarin can improve brain edema in SAE rats,reduce EB exudation,and inhibit the expression of MMP-9 and increase the expression of Claudin-5.Conclusion: Our research shows that puerarin may improve sepsis related encephalopathy and play a role in brain protection by inhibiting NLRP3/Caspase-1/ GSDMD mediated classical pathway of pyroptosis and reducing blood brain barrier damage.It may provide a new strategy for the treatment of SAE.