| Objective:To observe the effect of Qingnao Dripping Pill on inflammatory response in acute cerebral infarction,and clarify that Qingnao Dripping Pill exerts anti-inflammatory and neuroprotective effects by regulating miR-223 mediated NLRP3 inflammasomes.Methods:1.Seventy five male SD rats were selected.They were injected into the lateral ventricle with an antagonist of miR-223(antagomiR-223),and then the MCAO model was induced by thread occlusion.After 1.5 h of ischemia and 24 h of reperfusion,they were randomly divided into five groups:sham group(sham),MCAO model group(MCAO),Qingnao Dripping Pill group(QNDP),miR-223 antagonist group(antagomiR-223),Qingnao dripping pill+miR-223 antagonist group(QNDP+antagomiR-223).2.Garcia JH scoring was used to measure the degree of neurological impairment in rats;Observe the volume of cerebral infarction in rats using TTC staining;Measure the brain water content of rats using the dry wet weight.Take the ischemic cortex of rats for detection,and measure the expression of miR-223 in rats using RT-PCR;Western blot was used to detect the expression of NLRP3,ASC,and c-caspase-1 proteins in rats;The expression of NLRP3 and microglia was observed by immunofluorescence;ELISA method for detecting IL-10,IL-6,and IL-1β and IL-18 levels in rats.3.The mouse BV2 microglia was selected,and Lentivirus sh-miR-223 was added to the cells for transfection and incubation.After successful transfection,OGD model was established.They were divided into five groups:control group(con),oxygen glucose deprivation group(OGD),Qingnao Dripping Pill group(QNDP),miR-223 silent expression group(sh-miR-223),and Qingnao Dripping Pill+miR-223 silent expression group(QNDP+sh-miR-223).4.The activity of microglia was detected by CCK8;the mortality of microglia was measured by LDH.The expression of lncRNA MEG3 and miR-223 in microglia was detected by RT-PCR;the protein expressions of NLRP3,ASC and c-caspase-1 in microglia were detected by Western blot.5.Detection of Luciferase activity:lncRNA MEG3 binds to miR-223.Results:1.The neuroprotective effect of Qingnao Dripping Pill on MCAO rats.Compared with the sham group,the MCAO group showed a significant decrease in neurological scores(P<0.01),while the volume of cerebral infarction significantly increased(P<0.01)and brain water content increased(P<0.05),with statistically significant differences;compared with the MCAO group,the QNDP group showed a significant increase in neurological scores(P<0.01),a decrease in cerebral infarction volume and brain water content(P<0.01),the antagomiR-223 group showed a significant decrease in neurological scores(P<0.01),and an increase in cerebral infarction volume and brain water content(P<0.01).The difference was statistically significant.Compared with the QNDP group,the QNDP+antagomiR-223 group showed a significant decrease in neurological scores(P<0.01),and a significant increase in cerebral infarction volume(P<0.01)and an increase in water content(P<0.05),with statistically significant differences.2.Qingnao Dripping Pill regulate the expression of miR-223,NLRP3 inflammasome and microglia in MCAO rats’ ischemic cortex.Compared with sham group,the expression of miR223 in MCAO group significantly increased(P<0.01),the expression of NLRP3 significantly increased(P<0.01),ASC protein increased(P<0.05),c-caspase-1 rotein significantly increased(P<0.01),and the expression of NLRP3 and microglia significantly increased(P<0.01);compared with MCAO group,the expression of miR-223 in QNDP group significantly increased(P<0.01),the expression of NLRP3 significantly decreased(P<0.01),ASC and ccaspase-1 protein decreased(P<0.05),the expression of NLRP3 and microglia significantly decreased(P<0.01),the expression of miR-223 in antiagomiR-223 group decreased(P<0.05),the expression of NLRP3 increased(P<0.05),ASC and c-caspase-1 protein significantly increased(P<0.01),and the expression of NLRP3 and microglia significantly increased(P<0.01).The difference is statistically significant.Compared with QNDP group,the expression of miR-223 in QNDP+antiagomiR-223 group was significantly reduced(P<0.01),the expression of NLRP3,ASC and c-caspase-1 protein was increased(P<0.01),and the expression of NLRP3 increased(P<0.05)and microglia was significantly increased(P<0.01).3.The regulatory effect of Qingnao Dripping Pill on inflammatory cytokines in the ischemic cortex of MCAO rats.Compared with the sham group,MCAO group rats showed IL6,IL-1β and IL-18 significantly increased(P<0.01),while the level of IL-10 significantly decreased(P<0.01),with a statistically significant difference;compared with the MCAO group,the QNDP group showed IL-6,IL-1β and IL-18 reduced(P<0.01),while the content of IL-10 was significantly increased(P<0.01);the levels of IL-6,IL-1β and IL-18 in the antagomiR223 group of rats were significantly increased(P<0.01)while the content of IL-10 decreased(P<0.05),with a statistically significant difference;compared with the QNDP group,the QNDP+antagomiR-223 group showed IL-6,IL-1β and IL-18 significantly increased(P<0.01),while the content of IL-10 significantly decreased(P<0.01),with statistically significant differences.4.Protective effect of Qingnao Dripping Pill on OGD stimulated BV2 microglia.Compared with con group,the activity of microglia in OGD group was significantly lower(P<0.01),and LDH was significantly higher(P<0.01);compared with OGD group,the activity of microglia in QNDP group increased(P<0.05)and LDH decreased(P<0.05),with a statistically significant difference;compared with OGD group,the activity of microglia in shmiR-223 group was significantly lower(P<0.01),and LDH was higher(P<0.05);compared with QNDP group,the activity of microglia in QNDP+sh-miR-223 group was significantly lower(P<0.01),and LDH was significantly higher(P<0.05).5.Qingnao Dripping Pill regulates the expression of lncRNAMEG3 and miR-223 in BV2 microglia stimulated by OGD.Compared with con group,the expression of lncRNA MEG3 increased significantly(P<0.01)and miR-223 in microglia of OGD group increased(P<0.05);Compared with OGD group,the expression of lncRNA MEG3 in microglia of QNDP group decreased(P<0.01),and the expression of miR-223 increased(P<0.05),with a statistically significant difference;Compared with OGD group,the expression of lncRNA MEG3 in microglia of sh-miR-223 group had no significant change(P>0.05),while the expression of miR-223 in microglia of sh-miR-223 group was downregulated(P<0.01),with a statistically significant difference;Compared with QNDP group,there was no significant change in the expression of lncRNA MEG3 in microglia of QNDP+sh-miR-223 group(P>0.05),while the expression of miR-223 in microglia of QNDP+sh-miR-223 group decreased(P<0.05),which was statistically significant.6.Qingnao Dripping Pill regulates the expression of NLRP3 inflammasome in BV2 microglia stimulated by OGD.Compared with con group,the expression of NLRP3 and ASC protein in microglia of OGD group increased(P<0.05),and c-caspase-1 protein in microglia of OGD group significantly increased(P<0.01);Compared with OGD group,the protein expressions of NLRP3,ASC and c-caspase-1 in microglia of QNDP group decreased significantly(P<0.05);Compared with OGD group,the protein expressions of NLRP3 in microglia of sh-miR-223 group were significantly upregulated(P<0.01),ASC and c-caspase1 in microglia of sh-miR-223 group were upregulated(P<0.05),with statistically significant difference;Compared with QNDP group,the expression of NLRP3,ASC and c-caspase-1 protein in microglia of QNDP+sh-miR-223 group increased significantly(P<0.05).7.Expression of luciferase activity.Compared with the miR-223-3’UTR-NC+lncRNA MEG3 group,the expression of luciferase relative activity in the miR-223-3’UTR+lncRNA MEG3 group was significantly lower(P<0.01).Conclusion:1.Qingnao Dripping Pill significantly reduced the volume of cerebral infarction in MCAO rats,increased the activity of BV2 microglia in OGD,and had neuroprotective effects,providing scientific basis for the clinical application of heat clearing and blood activating method.2.Qingnao Dripping Pill can downregulate the expression of NLRP3 inflammasome and reduce the inflammatory reaction of microglia by upregulating the expression of miR-223 after acute cerebral infarction.The mechanism may be related to the regulation of lncRNAMEG3. |
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