Fig 1.
Schematic diagram illustrating the mechanism underlying the activation of the NLRP3 inflammasome by the ATP/P2X7R-Pannexin-1 signaling pathway in CD.
Note: ATP/P2X7R-Pannexin-1 signaling pathway induces the activation of NF-κB, which causes the transcription of NLRP3 mRNA. When NLRP3 is activated, ASC recruits pro-caspase-1 to assemble into a large cytosolic complex via the CARD domain, triggering the activation of caspase-1. Subsequently, active caspase-1 cleaves pro-IL-1β and pro-IL-18 and then releases biologically active mature IL-1β and IL-18, which induce inflammatory and autoimmune conditions including CD.
Fig 2.
Illustration of herb-partitioned moxibustion.
Note: HPM treatment was administered by placing a moxa cone on the top of an herbal cake at the Tianshu (ST25, bilateral) and Qihai (CV6) points and igniting it.
Table 1.
Primer sequences.
Fig 3.
CMDIs and colon length in each group.
Note: (A) CMDIs in each group. (B) Colon length in each group. (C) Colon images in each group. aP < 0.01, bP < 0.05 vs the NG; cP<0.01, dP<0.05 vs the MG. NG: normal group; MG: model group; HPMG: herb-partitioned moxibustion group; BBG-G: brilliant blue G group; SMG: sham moxibustion group. Data are presented as the Median (P25, P75) in (A) and () in (B), n = 8.
Fig 4.
Morphological observation and histopathological scores of colon tissues from each group.
Note: aP < 0.01 vs the NG; dP<0.05 vs the MG. NG: normal group; MG: model group; HPMG: herb-partitioned moxibustion group; BBG-G: brilliant blue G group; SMG: sham moxibustion group. The red arrow indicates inflammatory cell infiltration. Data are presented as the Median (P25, P75). Scale bar: 100 μm, n = 8.
Fig 5.
ATP content and the protein and mRNA expression of P2X7R protein in colon tissues from each group.
Note: (A) The ATP content in colon tissues in each group. (B) Western blot analysis of P2X7R protein in colons in each group. (C) RT-qPCR analysis of P2X7R mRNA in colons in each group. aP < 0.01 vs the NG; cP<0.01 vs the MG; eP<0.01,fP<0.05 vs the HPMG; gP<0.01 vs the BBG-G. NG: normal group; MG: model group; HPMG: herb-partitioned moxibustion group; BBG-G: brilliant blue G group; SMG: sham moxibustion group. Data are presented as the (), n = 8.
Fig 6.
The protein and mRNA expression of Pannexin-1 in colon tissues from each group.
Note: (A~E) Immunofluorescence of Pannexin-1 in colon tissues in each group with Pannexin-1 (red) and nucleus (blue). (F) IOD analysis of Pannexin-1 in colon tissues in each group. (G) RT-qPCR analysis of Pannesin-1 mRNA in colon tissues in each group. aP < 0.01 vs the NG; cP<0.01 vs the MG; eP<0.01,fP<0.05 vs the HPMG; gP<0.01 vs the BBG-G. NG: normal group; MG: model group; HPMG: herb-partitioned moxibustion group; BBG-G: brilliant blue G group; SMG: sham moxibustion group. Data are presented as the (). Scale bar: 100 μm, n = 8.
Fig 7.
Images of NF-κBp65 (brown) immunostaining and corresponding IOD analysis of NF-κBp65 expression in colon tissues from each group.
Note: aP < 0.01 vs the NG; cP<0.01 vs the MG; gP<0.01 vs the BBG-G. NG: normal group; MG: model group; HPMG: herb-partitioned moxibustion group; BBG-G: brilliant blue G group; SMG: sham moxibustion group. Data are presented as the (). Scale bar: 100 μm, n = 8.
Fig 8.
Protein expression of p-NF-κBp65 and mRNA expression of NF-κBp65 in colon tissues from each group.
Note: (A) Western blot analysis of p-NF-κBp65 protein in colon homogenate in each group. (B) RT-qPCR analysis of NF-κBp65 mRNA in colon tissues in each group. aP < 0.01, bP<0.05 vs the NG; cP<0.01 vs the MG; eP<0.01,fP<0.05 vs the HPMG; gP<0.01 vs the BBG-G. NG: normal group; MG: model group; HPMG: herb-partitioned moxibustion group; BBG-G: brilliant blue G group; SMG: sham moxibustion group. Data are presented as the (), n = 8.
Fig 9.
Protein expression of NLRP3, ASC, and caspase-1 in colon tissues from each group.
Note: (A) Western blot analysis of NLRP3 protein in colon homogenate in each group. (B) Western blot analysis of ASC protein in colon homogenate in each group. (C) Western blot analysis of caspase-1 protein in colon homogenate in each group. aP < 0.01, bP <0.05 vs the NG; cP<0.01, dP<0.05 vs the MG; eP<0.01,fP<0.05 vs the HPMG; gP<0.01, hP<0.05 vs the BBG-G. NG: normal group; MG: model group; HPMG: herb-partitioned moxibustion group; BBG-G: brilliant blue G group; SMG: sham moxibustion group. Data are presented as the (), n = 8.
Fig 10.
Protein expression of IL-1β and IL-18 in colon tissues from each group.
Note: (A) Western blot analysis of IL-1β protein in colon homogenate in each group. (B) Western blot analysis of IL-18 protein in colon homogenate in each group. aP < 0.01, bP <0.05 vs the NG; dP<0.05 vs the MG; fP<0.05 vs the HPMG; hP<0.05 vs the BBG-G. NG: normal group; MG: model group; HPMG: herb-partitioned moxibustion group; BBG-G: brilliant blue G group; SMG: sham moxibustion group. Data are presented as the (), n = 8.