| Objective:By targeting NLRP3 inflammasome signaling pathway,to investigate the mechanism of herb-partitioned moxibustion（HPM）in treating Crohn’s disease（CD）and to illustrate the action mechanism of HPM in regulating the activation of NLRP3inflammasome signaling pathway,for providing scientific evidence for elucidating the mechanism of HPM for CD.Method:1.Male Sprague-Dawley（SD）rats were randomly divided into 4 groups:normal group,model group,herb-partitioned moxibustion group,and sham herb-partitioned moxibustion group.The rat model of CD were developed by TNBS（enema）referring to the Morris method,which is 5%TNBS and 50%alcohol mixed at 2:1 in volume.The NG did not receive modeling or any intervention.After the model was established,the MG did not receive any intervention;the Mox G received HPM treatment on Tianshu,Qihai acupoints;the SMG received same treatment as Mox G without lighting the cones.Examing and analyzing the DAI,length of colon and CMDI.Histopathological injury in colon tissues were observed by HE staining under light microscope and score it.The concentrations of interleukin（IL）-1βand IL-18 in rat serum were detected by ELISA.The protein expressions of NLRP3（NOD-like receptor pyrin domain containing 3）,ASC（apoptosis-associated speck-like protein containing a CARD）,caspase-1 and IL-1βin rat colon were detected by immunohistochemistry.The protein expression of IL-18 in rat colon was detected by Western blot.The NLRP3,ASC,caspase-1 m RNAs were detected by RT-q PCR.2.Male SD rats were were randomly divided into 5 groups:normal group,model group,herb-partitioned moxibustion,brilliant-blue G group,and sham HPM group.The BBG-G received intraperitoneal injection of BBG,and the other groups were treated in the same way as before.Examing and analyzing the DAI,length of colon,CMDI and histopathological injury score.The concentration of ATP in rats’colon was deteced by Chemiluminescence method.The protein expressions of NLRP3,ASC,caspase-1,P2X7R,p-NF-κBp65,IL-1βand IL-18 in rats’colon were detected by Western blot.The protein expression of NF-κBp65 in rats’colon was detected by IHC.The protein expression of Pannexin-1 in rats’colon was detected by immunofluorescence（IF）.The P2X7R,pannexin-1,NF-κBp65 m RNA in rats colon were detected by RT-q PCR.3.Male SD rats were randomly divided into 5 groups:normal group,model group,herb-partitioned moxibustion group,NAC group,and sham HPM group.Rats of NACG received NAC by enema,and the other groups were treated as above.Examing and analyzing the DAI,length of colon,CMDI,and histopathological injury score.The concentration of ROS in rats’colon was detected by fluorescent staining method.The protein expression of NLRP3,ASC,caspase-1,TXNIP,IL-1β,and IL-18were detected by Western blot.The protein expression of TRX,TXNIP and co-expression of TXNIP/NLRP3in rats’colon were detected by IF.The expression of TXNIP,TRX m RNA in rats’colon were detected by RT-q PCR.Results:1.The regulation of HPM on colonic NLRP3 inflammasome signaling pathway and it downstream molecular（IL-1βand IL-18）in CD ratsCompared with the NG,the DAI,CMDI,and histopathological injury score rose significantly in MG and SMG（P<0.01）.Compared with the NG,the length of colon was shorten significantly in MG and SMG（P<0.01）.Compared with the MG,the DAI,CMDI and histopathological injury score decreased significantly in Mox G（P<0.05）.And HE stain result showed severe colonic lesion with inflammation and ulcers in MG and SMG’s rats.Compared with the NG,the concentrations of IL-1βand IL-18 in rats’serum were significantly increased in the MG,Mox G and SMG（P<0.01）.Compared with the MG and SMG,the concentrations of IL-1βand IL-18 in rats’serum were significantly decreased in the Mox G（P<0.05）.Compared with the NG,the expressions of NLRP3,ASC,caspase-1,IL-1βand IL-18 in rats’colon were significantly increased in the MG and SMG（P<0.01）.Compared with the MG,the expressions of NLRP3,ASC,caspase-1,IL-1βand IL-18 in rats’colon were significantly decreased in the Mox G（P<0.05）.Compared with the NG,the m RNA expressions of NLRP3,ASC and caspase-1 in rats’colon were significantly increased in the MG and SMG（P<0.01）.Compared with the MG and SMG,the m RNA expressions of NLRP3,ASC and caspase-1 in rats’colon were significantly decreased in Mox G（P<0.05）.2.The effect of HPM on the upstream regulating pathways of colonic NLRP3inflammasome in CD rats1)The effect of HPM on the ATP/P2X7R-Pannexin-1 signaling pathway:Compared with the NG,the level of ATP was increased significantly in the MG,Mox G and SMG（P<0.01）.Compared with the MG and SMG,the level of ATP was significantly decreased in the Mox G and BBG-G（P<0.01）.Compared with the NG,the protein expressions of P2X7R,Pannexin-1,NF-κBp65,p-NF-κBp65,NLRP3,ASC and IL-1βin rats’colon were significantly increased in the MG and SMG（P<0.05）.Compared with the MG,the protein expressions of P2X7R,Pannexin-1,NF-κBp65,p-NF-κBp65,NLRP3,ASC,caspase-1,and IL-1βin rats’colon were significantly decreased in the Mox G and BBG-G（P<0.05）.Compared with the NG,the m RNA expressions of P2X7R,Pannexin-1 and NF-κBp65 in rats’colon were significantly increased in the MG,Mox G and SMG（P<0.01）.Compared with the MG and SMG,the m RNA expressions of P2X7R,Pannexin-1 and NF-κBp65 in rats’colon were significantly decreased in both Mox G and BBG-G（P<0.05）.2)The effect of HPM on the ROS/TXNIP-NLRP3 signaling pathway:Compared with the NG,the level of ROS in rats’colon was significantly increased in the MG,Mox G,NACG and SMG（P<0.05）.Compared with the MG and SMG,the level of ROS in rats’colon was significantly decreased in the Mox G and NACG（P<0.05）.Compared with the NG,the protein expressions of TXNIP,TRX,NLRP3,ASC,caspase-1 and IL-1βwere significantly increased in the MG and SMG（P<0.05）.Compared with the MG and SMG,the protein expressions of TXNIP,TRX,NLRP3,caspase-1 and IL-1βwere significantly decreased in the Mox G and NACG（P<0.05）.Compared with the NG,the m RNA expressions of TXNIP and TRX were significantly increased in the MG,Mox G,NACG and SMG（P<0.01）.Compared with the MG and SMG,the m RNA expressions of TXNIP and TRX were significantly decreased in the Mox G and NACG（P<0.01）.Conclusion:1.HPM can improve the CD rats’histopathological injury,relieve inflammation,and decrease the DAI.2.HPM can downregulate the activation of NLRP3 inflammasome（NLRP3,ASC,caspase-1）,as well as the expressions of it down-stream inflammatory factors IL-1βand IL-18.Inhibiting the over-activated NLRP3 inflammasome may be one of the mechanisms of HPM in treating CD.3.HPM can decrease the level of ATP in rats’colon,so as to downregulate the protein and m RNA expressions of P2X7R,Pannexin-1 and NF-κBp65.It is indicated that HPM may play its therapeutic effect via regulating the ATP/P2X7R-Pannexin-1pathway in CD rats’colon to inhibit the abnormal activation of downstream NLRP3inflammasome.4.HPM can decrease the level of ROS in rats’colon,downregulating the protein and m RNA expressions of TXNIP and TRX.It is suggested that HPM may play its therapeutic role by regulating the colonic ROS-TXNIP signaling pathway to inhibit the over-activated downstream NLRP3 inflammasome. |
