Cistanche Deserticola Polysaccharides Attenuates Diabetic Nephropathy in Mice By Affecting The Intestinal Flora And Inhibiting The Toll-Like Receptor 4/Nuclear Factor-κB Signaling Pathway Ⅱ

2.5 Effect of CDPs on colon pathology in C57BL/6J mice

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As shown in Figure 2, H&E staining revealed that the colon tissue structure of normal mice was normal, the crypts were neatly and tightly arranged, the number was large, and the epithelial cells had no obvious degeneration and shedding. In contrast, DN mice showed obvious shedding of colon epithelial cells, infiltration of inflammatory cells, damage to crypt cells, disappearance of intestinal glandular structure, and increased permeability. After 4 weeks of CDPs intervention, the above pathological conditions recovered to varying degrees.

AB-PAS staining results showed that compared with the Control group, the number of unemptied goblet cells in the colon of the Model group mice was significantly reduced, and the secretion of acidic mucus protein was significantly increased; the number of unemptied goblet cells in the colon of the Control group was the largest; compared with the Model group, the secretion of colonic acidic mucus protein in the CDPs-H group was reduced.

Fig. 2 Effect of CDPs on colonic histopathologic changes in DN mice as examined by H&E and AB-PAS staining

2.6 Effect of CDPs on intestinal flora of C57BL/6J mice

The effects of CDPs on biochemical indicators and pathological results of DN mice were comprehensively analyzed, and the colon contents of mice in the CDPs-H group with the best overall efficacy were selected for intestinal flora research.

2.6.1 Amplicon sequence variant (ASV) distribution Venn diagram As shown in Figure 3, compared with the Control group, the relative abundance of intestinal flora species in the Model group mice decreased, and the relative abundance improved after CDPs treatment.

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2.6.2 Alpha diversity analysis As shown in Table 6, compared with the Control group, the Chao1 index of the intestinal flora of the Model group mice decreased. After CDPs intervention, the Chao1 index increased, but there was no statistical significance, indicating that CDPs may change the intestinal flora structure by increasing the total number of species. The Coverage index is close to 1, indicating that the sequencing results can basically reflect the true situation of the sample. There was no statistically significant difference in Shannon, Simpson, Coverage, and Pielou_e index among the groups, indicating that the species richness and diversity of the environmental communities in each group were good, and the colony coverage was consistent and evenly distributed.

Fig. 3 Venn diagram of ASV distribution

Table 6 Alpha-diversity indices of intestinal microbiota in mice in the control, model and CDPs groups (n = 6)

2.6.3 Beta diversity analysis

As shown in Figure 4, through Beta diversity analysis, principal component analysis (PCA) can distinguish three groups of samples in three-dimensional space, indicating that there are significant differences in the intestinal flora of these three groups of samples. The Model group and the Control group are separated along the PC2 axis, and the CDPs-H group is between the Model group and the Control group, close to the Control group.

Fig. 4 Beta diversity of intestinal flora in mice in the control, model and CDPs groups (n = 6)

2.6.4 Effects of CDPs on the intestinal flora of C57BL/6J mice

2.6.4.1 Phylum level

As shown in Figure 5, compared with the Control group, the relative abundance of Firmicutes in the Model group increased significantly, the relative abundance of Bacteroidota and Verrucomicrobia decreased significantly, and the relative abundance ratio (B/F) of Bacteroidetes to Firmicutes decreased. Compared with the Model group, CDPs significantly increased the relative abundance of Bacteroidetes and Verrucomicrobia, decreased the relative abundance of Firmicutes, and increased the B/F value, which was closer to the B/F value of the Control group.

Fig. 5 Bar chart of the relative abundance of intestinal flora at the phylum level (n = 6)

2.6.4.2 Genus level

The histogram of species abundance composition at the top 30 genera levels (Figure 6) shows that there are differences in the intestinal flora of mice in the Model group and the CDPs-H group. There are 16 different bacterial genera between the Model group and the Control group; there are 8 different bacterial genera between the CDPs-H group and the Model group; 5 bacterial genera in the overlapping area may be the bacterial genera that CDPs can improve diabetic nephropathy (Figure 7). As can be seen from Table 7, at the genus level in the mouse intestinal flora, compared with the Control group, the relative abundance of Allobaculum in the Model group was significantly reduced, and the relative abundance of Roseburia, Romboutsia, Ruminococcus_torques_group and RF39 (P<0.05) was increased. ; Compared with the Model group, the relative abundance of Roseburia, Romboutsia, Ruminococcus_torques_group and RF39 (P＜0.05) decreased in the CDPs-H group; the relative abundance of Allobaculum increased (P＜0.05).

Fig. 6 Bar chart of the relative abundance of intestinal flora at the genus level (n = 6)

Fig. 7 Venn diagram of intestinal flora at the genus level (n = 6)

Table 7 Effects of CDPs on the relative abundance of differential gut microbial genera between the control, model and CDPs groups (n = 6)

2.6.4.3 Effect of CDPs on the expression of colonic ZO-1 and Occludin

As shown in Figure 8 and Table 8, compared with the Control group, the relative expression levels of ZO-1 and Occludin proteins in the colon tissue of mice in the Model group were significantly reduced (P < 0.01); compared with the Model group, CDPs intervention at each dose could The relative expression levels of ZO-1 and Occludin proteins in colon tissue were significantly increased (P<0.01).

Fig. 8 Expression of ZO-1 and occludin in colon tissue of mice from each group (n = 3)

Table 8 Relative expression levels of ZO-1 and occludin in colon tissue of mice from each group (n = 3)

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2.7 Effect of CDPs on renal TLR4/NF-&kappa;B signaling pathway protein expression

As shown in Figure 9 and Table 9, compared with the Control group, the relative expression of TLR4 protein in the kidney tissue of mice in the Model group and the ratios of p-NF-&kappa;Bp65/NF-&kappa;B p65 and p-I &kappa;B&alpha;/I &kappa;B&alpha; were significantly increased. High (P<0.01); compared with the Model group, the relative expression of TLR4 protein and p-NF-&kappa;Bp65/NF-&kappa;B p65 and p-I &kappa;B&alpha;/I &kappa;B&alpha; in the kidney tissue of mice in the CDPs-M and CDPs-H groups The ratios were significantly reduced (P＜0.01, P＜0.05).

Fig. 9 Western blot analysis of protein expression related to the TLR4/ NF-&kappa;B signaling pathway in kidney tissue of mice from each group (n = 3)

Table 9 Relative expression levels of TLR4/NF-&kappa;B signaling pathway-related proteins in kidney tissue of mice from each group (n = 3)