NLRP3-Related Inflammatory Reaction In Spinal Cord After Cerebral Ischemia In Rats And The Regulatory Effect Of Ginsenoside Rg1

Objective: Using a rat model of middle cerebral artery occlusion(MCAO),ginsenoside Rg1(Rg1)was administered to investigate changes in differential genes and inflammation-associated factors such as NLRP3 inflammasomes in the spinal cord after cerebral ischemia in rats,and revealed the anti-inflammatory effects of Rg1,providing a theoretical basis for preventing secondary inflammatory responses in the spinal cord after cerebral ischemia.Methods: A total of 59 adult SD male rats were randomly divided into the group of sham surgery control(Sham group,n=22),administration of saline after middle cerebral artery occlusion(MCAO group,n=22)and ginsenoside Rg1 therapy after middle cerebral artery occlusion(Rg1 group,n=15).The rat MCAO model was replicated by isolating the right middle cerebral artery in rats,which was not isolated in Sham group;the next day after successful modeling,Rg1(10 mg/ml)was injected intraperitoneally at a dose of 4 mg/100 g in the Rg1 group,and the same amount of saline was given in the MCAO group for 3 consecutive days.Behavioral studies were performed to examine motor function in each group after surgery.Rats in each group were harvested on postoperative day 3.Rats were killed and cervical enlargement of their cervical spinal cord was removed,changes in genes,microglia,NLRP3 inflammasome,and inflammatory cytokines were determined respectively by transcriptome sequencing,q RT-PCR,immunofluorescence staining,and Western blot.BM SPSS Statistics 21.0 statistical software was used to carry out one-way ANOVA and pairwise comparison of the data.The data was expressed by mean ± standard deviation,and the difference was statistically significant when P <0.05.Results: 1.The behavioral results showed that the left forepaw of Sham rats was spontaneously fully relaxed at the contact level;the left forepaw of MCAO rats was limited in its relaxation function;and the left forepaw of Rg1 rats was slightly improved.Climbing footprint analysis revealed clear,consistent footprints in Sham rats.Foot tracks in the MCAO group are chaotic and drawn.In the Rg1 group,the footprint tended to be normal.Strab testing showed that the Sham group stabilized the limbs at a 70 degrees slope;the MCAO group stabilized only at a 60 degrees slope,which was significantly lower than the Sham group(P <0.01);and the Rg1 group had an increase in angles on days 1 and 3 compared with the MCAO group(P <0.01),but did not return to normal levels.2.Transcriptome sequencing revealed a total of 1556 differentially expressed genes in the MCAO versus Sham group,913 up-regulated and643 down-regulated.Key items in Functional Enrichment were chemokine signaling and leukocyte transendothelial migration.NLRP3,Psycard,caspase-8,Nlrc4,Mefv,and Syk were upregulated in the MCAO spinal cord compared to Sham.3.q RT-PCR showed higher expression of NLRP3,Pycard,caspase-8,Nlrc4,Mefv,and Syk in the MCAO spinal cord compared with the Sham group(P <0.01).4.Immunofluorescence staining showed that small glial cells were present in the spinal cord of Sham rats with small and long processes;microglia in MCAO group had enlarged cell bodies and shortened prominence.Microglia in the Rg1 group had a smaller corpus callosum and increased perisomatic small processes.5.Western blot analysis revealed a significant increase in NLRP3,ASC,caspase-1,TNF-α,and IL-1β in the MCAO spinal cord compared with Sham(P <0.05),and a decrease in NLRP3,ASC,caspase-1,TNF-α,and IL-1β in the Rg1 group compared with Sham(P <0.05),but no return to Sham levels.Conclusions: 1.Following cerebral ischemia in rats,secondary damage to the spinal cord occurs,and a large number of genes are abnormally expressed and closely related to biological processes and signaling pathways such as inflammation,apoptosis,and metabolic abnormalities.2.In rats,spinal cord microglia and NLRP3 inflammasomes are activated and the expression of inflammatory cytokines is increased after cerebral ischemia.3.Ginsenoside Rg1 downregulates NLRP3 inflammasome and inflammatory cytokine expression in the spinal cord of rats after cerebral ischemia,inhibits microglial activation,and protects from impaired spinal cord neurogenesis.