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

Figure 1 Ultra-performance liquid chromatography tandem mass spectrometry extracted ion chromatograms of Runchangningshen paste. A: Positive ion modes; B: Negative ion modes.

Figure 2 Runchangningshen paste relieved the inhibitory effect of functional constipation on the defecation function. A: Measurement of body weight (n = 6); B: Measurement of food intake (n = 6); C-E: Measurement of the 24-h fecal index (n = 6) (C: Fecal number; D: Fecal weight; E: Water content of feces); F and G: Measurement of gastrointestinal motility. Rats were intragastrically administered the black solid paste and sacrificed 30 min later. The stomach was removed, and the weights of the entire stomach and empty stomach were measured. The intestine was removed, and the rate of the black solid paste propulsion was calculated (n = 6) (F: Gastric emptying rate; G: Intestinal propulsion rate test). 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.

Figure 3 Runchangningshen paste alleviated the impairment effect of functional constipation on the colonic mucosal layer and the mucin production. A: Representative image of hematoxylin and eosin staining (× 200 magnification). The yellow arrows indicate the mucosal epithelial cells, the red circles indicate the goblet cells, the green zones indicate the colonic crypts, the orange boxes indicate the fibrous tissue, and the blue circles indicate the glands; B: Representative image of Alcian blue-periodic acid-schiff staining (× 200 magnification). Purple-pink indicates mucin. FC: Functional constipation model 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; NC: Normal control group.

Figure 4 Runchangningshen paste improved the inhibition effect of functional constipation on autophagy and mucin exocytosis in goblet cells. The representative picture of the ultrastructure of the goblet cells by the transmission electron microscope (× 3400 magnification). The blue area and dots indicate the mucins, the red arrows indicate the autophagosome, and the yellow arrows indicate the damaged mitochondria. FC: Functional constipation model 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; NC: Normal control group.

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.

Figure 6 Runchangningshen paste changed the gut mucosal microbiota diversity in functional constipation rats. A: Pan curve; B: Core curve; C: Rank-Abundance curves; D-G: Alpha diversity analysis. The y-axis represents the operational taxonomic unit (OTU) level corresponding to each group based on the different indexes (n = 6) (D: Simpson index analysis; E: Shannon index analysis; F: Chao index analysis; G: Ace index analysis); H-M: Beta diversity analysis. The x-axis and y-axis represent two selected principal component (PC1 and PC2) axes, while the percentages indicate the explanatory values of the principal components regarding the differences in sample composition. Stress is used to assess the quality of the Non-metric multidimensional scaling analysis (NMDS) analysis results. Different colors or shapes represent different samples or groups. The closer the sample points or shapes are, the more similar the species composition of the two or two groups of samples becomes (n = 6) [H: Principal coordinates analysis (PCoA); I: Principal component analysis (PCA); J: NMDS analysis; K: Partial least squares discriminant analysis (PLS-DA); L: ANOSIM analysis; M: Kruskal-Wallis; H test analysis). All data were shown as the mean ± SD. P values were calculated using the ANOSIM test. ANOVA: Analysis of variance; FC: Functional constipation model 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; NC: Normal control group.

Figure 7 Runchangningshen paste changed the composition of gut mucosal microbiota at the multispecies level in functional constipation rats. A: Operational taxonomic unit analysis: Venn diagrams; B: Community heatmap analysis at the phylum level between normal control group (NC), functional constipation model group (FC), runchangningshen paste group (RCNSP), and lactulose group (LAC) group; C: Diagram of intergroup comparisons of NC, FC, and high RCNSP (HRCNSP) at the phylum level (n = 6); D: Diagram of intergroup comparison of NC and FC at the phylum level (n = 6); E: Diagram of intergroup comparison of FC and HRCNSP at the phylum level (n = 6); F: Community heatmap analysis at the family level between NC, FC, RCNSP, and LAC; G: Diagram of intergroup comparisons of NC, FC, and HRCNSP at the family level (n = 6); H: Diagram of intergroup comparison of NC and FC at the family level (n = 6); I: Diagram of intergroup comparison of FC and HRCNSP at the family level (n = 6); J: Community heatmap analysis at the genus level between NC, FC, RCNSP, and LAC; K: Diagram of intergroup comparisons of NC, FC, and HRCNSP at the genus level (n = 6); L: Diagram of intergroup comparison of NC and FC at the genus level (n = 6); M: Diagram of intergroup comparison of FC and HRCNSP at the genus level (n = 6). In graphs B, F, and J, the x-axis represents the group names, while the y-axis denotes the species names. The color gradient of the blocks illustrates the variations in abundance of different species within the groups, with the values represented by the color gradient displayed on the right side of the figure. In graphs C-E, G-I, and K-M, the x-axis shows the mean proportion or proportion difference of species in the sample, and the y-axis lists the species names or P values. The different colors of the bars denote distinct groups. The width of the bars reflects the magnitude of the species’ proportions. Data were expressed as mean ± SD. P values were calculated using Kruskal-Wallis H test or Wilcoxon rank-sum test. LRCNSP: Low dose of runchangningshen paste group; MRCNSP: Medium dose of runchangningshen paste group.

Figure 8 Runchangningshen paste modulated the mucosal microbiota composition to promote the defecation in functional constipation rats. A: Heatmap of the correlation between the differential microbiota at the phylum level and the indicators of the defecation function (n = 6); B: Heatmap of the correlation between the differential microbiota at the family level and the indicators of the defecation function (n = 6); C: Heatmap of the correlation between the differential microbiota at the genus level and the indicators of the defecation function (n = 6). Average was used for both clinical factors and species hierarchical clustering methods. We calculated the correlation between variables using the Spearman’scorrelation coefficient. The x-axis and y-axis represent clinical factors and species, respectively. The correlation coefficients (r values) and P values were obtained through calculations. The r values are displayed in the figure using different colors. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 9 Runchangningshen paste changed the mucosal microbiota function to promote mucin-2 secretion in functional constipation rats. A: Heatmap of Kyoto Encyclopedia of Genes and Genomes database (KEGG) functional enrichment of differential microbiota between normal control group (NC), functional constipation model group (FC) and high dose of runchangningshen paste (HRCNSP) groups at the pathway level 1 (n = 6); B: Heatmap of KEGG functional enrichment of differential microbiota between NC, FC and HRCNSP groups at the pathway level 2 (n = 6); C: Heatmap of KEGG functional enrichment of differential microbiota between NC, FC and HRCNSP groups at the pathway level 3 (n = 6); D: Orthologous groups of proteins database functional classification diagram of differential microflora among NC, FC, and HRCNSP groups (n = 6). In Figures A-C, the x-axis represents the group names, while the y-axis indicates the functional names at pathway levels 1/2/3. The color gradient of the blocks illustrates the variations in functional abundance across the groups, with the legend indicating the values represented by the color gradient. In Figure D, different colors represent different functional classifications, the x-axis represents the relative abundance of functional classifications, and the y-axis represents groups. COG: Cluster of Orthologous Groups.

Figure 10  Runchangningshen paste adjusted the relationship between the mucosal microbiota and NLRP6/autophagy pathway to promote mucin-2 secretion in functional constipation rats. A: Heatmap of the correlation between the differential microbiota at the phylum level and NLRP6/autophagy pathway (n = 6); B: Heatmap of the correlation between the differential microbiota at the family level and NLRP6/autophagy pathway (n = 6); C: Heatmap of the correlation between the differential microbiota at the genus level and NLRP6/autophagy pathway (n = 6). The average was used for both clinical factors and species hierarchical clustering methods. We calculated the correlation between variables using the spearman's correlation coefficient. The x-axis and y-axis represent clinical factors and species, respectively. The correlation coefficients (r values) and P values were obtained through calculations. The r values are displayed in the figure using different colors. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.