Characterization of Human ADSC
ADSC expressed all specific MSC markers CD73, CD105, CD90, CD44 and lacked expression of the hematopoietic markers CD34 and CD45. Differentiation to adipocytes, chondrocytes, and osteocytes was observed in ADSC (Fig. s1).
ADSC and Rg1 administration ameliorated DSS-induced colitis
As Fig. 1b shown, the body weights of the mice in the DSS groups were consistently reduced since the fifth day, while the mice in the ADSC + Rg1 groups and Rg1 groups mice showed a significant improvement in weight loss, although there was an apparent decrease in the ADSC groups (P < 0.05). In the colitis model, disease severity is typically associated with colon length shortening due to intestinal inflammation . The length of colon in the DSS groups were shorter than in the ADSC groups, Rg1 groups, and ADSC + Rg1 groups (p < 0.05).
Consistent with previous studies , H&E colon analysis (Fig. 1e) showed that DSS-induced colitis resulted in extended ulcerations, destroyed crypts and transmural inflammatory infiltration, with a barely complete mucosal structure. However, in mice treated with ADSC and Rg1, histological damage was ameliorated, as evidenced by a preserved mucosal architecture showing focal erosions and mild/moderate mucosa inflammatory infiltration. To evaluate the intestinal mucosal architecture and inflammatory infiltration, we used the histopathology score system to quantity inflammatory severity degree, and the mice treated with ADSC and Rg1 had a significantly lower score than the mice treated with DSS only (P < 0.05) (Fig. 1f).
ADSC and Rg1 treatment could alleviate colitis by regulating pro/anti-inflammatory cytokines
Pro-inflammatory cytokines play a crucial role in the progression of DSS-induced colitis . To explore whether ADSC and Rg1 treatment could alleviate colitis by regulating inflammation, we detected the cytokine expression in blood serum. As shown in Figure
.2a-h, the levels of inflammatory cytokines IL-6, IL-33, TNF-α, IL-1β and IL-17A in Rg1, ADSC, and ADSC + Rg1 groups decreased significantly and IL-10 increased compared with those in the DSS groups. Moreover, we found that the combined of ADSC + Rg1 groups can significantly inhibit the expression of inflammatory cytokines expression compared to Rg1 and ADSC alone groups (Fig. 2a, b, c, d, e, f, g, h). Therefore, Rg1 can enhance the effect of ADSC on DSS-induced colitis in a mouse model.
ADSC and Rg1 regulated Treg/Th17 balance to maintain intestinal homeostasis
Previous study has demonstrated that ADSC could inhibit Th17 response in T-cell-mediated autoimmune diseases. It has also been reported that ADSC could inhibit Treg/Th17 differentiation in DSS-induced colitis model mice. So we further compared the number of Th17 cells between groups. We found the number of Th17 cells were much higher in the DSS group than in the Rg1, ADSC, and ADSC + Rg1 groups (Fig. 3b, c, e). But rather, the percentage of Treg cells was remarkably higher in Rg1, ADSC, and ADSC + Rg1 groups compared with DSS groups. In summary, it indicated that Rg1 and ADSC administration selectively upregulated the frequency of Treg cells as well as downregulated the ratio of Th17 cells against DSS-induced colitis, improving the Treg/Th17 balance to maintain intestinal homeostasis. (Fig. 3a, d, f). Besides, the ADSC and Rg1 treated simultaneously showed better trends of recovering Treg/Th17 balance than ADSC and Rg1 groups alone.
ADSC and Rg1 treatment significantly altered the gut microbiota diversity and composition
Gut microbiota is an important factor in regulate intestinal inflammation . Therefore, we further compared the microbiota composition of five groups. The alpha diversity of bacterial communities was evaluated according to Shannon’s diversity index (Fig. 4a). The Shannon’s diversity index of ADSC and Rg1 showed no statistical difference with DSS groups (Fig. 4a, P > 0.05). The PCoA scatterplot revealed the clear clustering of gut bacterial communities between five groups (Fig. 4c). To compare the gut microbiota composition of the five groups, we then conducted a statistical analysis of gut microbiota at the genus level using the Kruskal–Wallis test. At the genera level, compared to DSS groups, the gut microbiota of ADSC, Rg1, and ADSC + Rg1 groups was characterized by an increased Rikenellaceae RC9 and ruminococcaceae UCG-013 level (Fig. 4d) and a lower ratio of Erysipelatoclostridium and Escherichia-shigella (Fig. 4d). Interestingly, the ratio of Rikenellaceae RC9, Ruminococcaceae UCG-013, Erysipelatoclostridium and Escherichia-shigella lever in ADSC + Rg1 groups were more similar to control groups. In addition, we also found that the effect of ADSC on gut microbiota disturbance induced by DSS was not significantly improved, there was even a worsening trend. However, Rg1 can restore the disturbed gut microbiota induced by DSS treated by ADSC.
We also identified significant changes in the multiple biological pathways of five groups (Fig. 4e). As shown in Fig. 4e, the fifteen modules in the five groups were involved in L-arginine degradation, superpathway of L-arginine, putrescine, and 4-aminobutanoate degradation, superpathway of methylglyoxal degradation, glyoxylate cycle, Citrate cycle (TCA cycle), L-methinonine biosynthesis, and heme biosynthesis, and methanogenesis from acetate. The overrepresentation of the L-arginine degradation pathway in DSS groups may be related to the high abundance of Escherichia-shigella, which has been shown to have a wide capacity for degrading polysaccharides in those samples. In all, the predication analysis indicated that gut dysbiosis may induce a disease-linked state through the interference of physiological metabolic functions.