[Regulatory effects of umbilical cord mesenchymal stem cells and their conditioned medium on gut microbiota of septic mice]

Abstract

Objective: To investigate and compare the regulatory effects of umbilical cord mesenchymal stem cells (MSC) and their conditioned medium (MSC-CM) on gut microbiota of septic mice.

Methods: Twenty-eight six-to-eight-week-old female C57BL/6J mice were randomly divided into sham operation group (Sham group), sepsis model group (CLP group), sepsis+MSC treatment group (CLP+MSC group) and sepsis+MSC-CM treatment group (CLP+MSC-CM group), with seven mice in each group. The septic mouse model was established by cecal ligation and puncture (CLP). In Sham group, CLP were not performed, and other operations were the same as CLP group. Mice in the CLP+MSC group and CLP+MSC-CM group received 0.2 mL 1×10⁶ MSC or 0.2 mL concentrated MSC-CM via intraperitoneal injection 6 hours after CLP, respectively. Sham group and CLP group were given 0.2 mL sterile phosphate buffer saline (PBS) via intraperitoneal injection. Histopathological changes were evaluated by hematoxylin-eosin (HE) staining and colon length. Levels of inflammatory factors in serum were detected by enzyme-linked immunosorbent assay (ELISA). Phenotype of peritoneal macrophages was analyzed by flow cytometry, and the gut microbiota was analyzed via 16S rRNA sequencing.

Results: Compared with Sham group, significant inflammatory injury in lung and colon was observed, and shorter colon was detected in CLP group (cm: 6.00±0.26 vs. 7.11±0.09), the level of inflammatory cytokine interleukin-1β (IL-1β) in serum was significantly increased (ng/L: 432.70±17.68 vs. 353.70±17.01), the proportion of F4/80⁺ peritoneal macrophages was increased [(68.25±3.41)% vs. (50.84±4.98)%], while the ratio of F4/80⁺CD206⁺ anti-inflammatory peritoneal macrophages was decreased [(45.25±6.75)% vs. (66.66±3.36)%]. The α diversity sobs index of gut microbiota was downregulated significantly (118.50±23.25 vs. 255.70±6.87), the structure of species composition was altered, and the relative abundance of functional gut microbiota related to transcription, secondary metabolites biosynthesis, transport and catabolism, carbohydrate transport and metabolism, and signal transduction were decreased significantly in CLP group (all P < 0.05). Compared with CLP group, upon MSC or MSC-CM treatment, the pathological injury in lung and colon was alleviated to varying extent, the length of colon was increased (cm: 6.53±0.27, 6.87±0.18 vs. 6.00±0.26), the level of IL-1β in serum was downregulated (ng/L: 382.10±16.93, 343.20±23.61 vs. 432.70±17.68), the ratio of F4/80⁺ peritoneal macrophages was decreased [(47.65±3.93)%, (48.68±2.51)% vs. (68.25±3.41)%], the ratio of F4/80⁺CD206⁺ anti-inflammatory peritoneal macrophages was increased [(52.73±5.02)%, (66.38±4.73)% vs. (45.25±6.75)%], and the α diversity sobs index of gut microbiota was increased (182.50±16.35, 214.00±31.18 vs. 118.50±23.25), and the effects of MSC-CM were more significant (all P < 0.05). At the same time, species composition of gut microbiota was rebuilt, and a tendency of increase in relative abundance of functional gut microbiota was observed upon MSC and MSC-CM treatment.

Conclusions: Both MSC and MSC-CM could alleviate inflammatory injury in tissues, and showed regulatory effects on gut microbiota in septic mouse model, moreover, MSC-CM exhibited superior advantages over MSC.