Runchangningshen paste activates NLRP6 inflammasome-mediated autophagy to stimulate colonic mucin-2 secretion and modulates mucosal microbiota in functional constipation

Figure 5 Runchangningshen paste attenuated the inhibition effect of functional constipation on the NLRP6/autophagy/mucin-2 pathway in colon tissue. A: The colon tissues were stained with the NLRP6 markers (green), LC3 markers (pink) and mucin-2 (MUC2) markers (red); B: NLRP6/LC3/MUC2 colocalization expression intensity by the Tissue Gnostics calculation (n = 3); C-H: The mRNA levels of NLRP6, Asc, Caspase1, interleukin (IL) 1β, IL18, and MUC2 were measured using reverse transcription quantitative PCR; I: The protein expression levels of NLRP6, Asc, Caspase1, IL1β, IL18 were analyzed by western blot; J-N: Density calculations for NLRP6, Asc, Caspase1, IL1β, IL18 (n = 6); O: The protein expression levels of LC3II, Beclin1, P62, and MUC2 were analyzed by western blot; P-S: Density calculations for LC3II, Beclin1, P62, and MUC2 (n = 6). All data were shown as the mean ± SD. P values were calculated using one-way analysis of variance followed by Fisher’s least significant difference test. ^aP < 0.0001 vs normal control (NC) group; ^bP < 0.05 vs functional constipation model (FC) group; ^cP < 0.01 vs FC group; ^dP < 0.001 vs FC group; ^eP < 0.0001 vs FC group. HRCNSP: High dose of runchangningshen paste group; LAC: Lactulose group; LRCNSP: Low dose of runchangningshen paste group; MRCNSP: Medium dose of runchangningshen paste group.