[Comparative study of the effect of different doses of human umbilical cord mesenchymal stem cells exosomes on intestinal barrier injury in severely burned rats].

Abstract

Objective: To explore the therapeutic efficacies of three different doses of human umbilical cord mesenchymal stem cell exosomes (hucMSC-EXO) on the injury of intestinal barrier structure and dysfunction in severely burned rats, and to identify the optimal dose of hucMSC-EXO for the repair of intestinal barrier injury. Methods: The hucMSC-EXO was isolated and identified by using an exosome extraction and purification kit. A total of 30 specific pathogen free (SPF) male Wistar rats (aged 6-8 weeks) were selected, and were randomly divided into five groups (n=6) using a random number table: sham group, burn group, burn+100 μg hucMSC-EXO group (Burn+EXO100), burn+200 μg hucMSC-EXO group (Burn+EXO200), and burn+400 μg hucMSC-EXO group (Burn+EXO400). The rats were immersed in 94 ℃ water, with the dorsal area exposed for 12 seconds and the ventral area for 6 seconds, to establish a 50% total body surface area (TBSA) third-degree burn model. The sham group rats were subjected under the same condition but with a 37 ℃ water bath. On day 1, 3, and 5 post-burn, the rats in sham group and burn group received an intraperitoneal injection of 0.5 ml phosphate buffered solution, and those in Burn+EXO100, Burn+EXO200, and Burn+EXO400 groups received intraperitoneal injections of 100, 200, and 400 μg/0.5 ml hucMSC-EXO, respectively. The activity of rats was observed and the weight was recorded daily. On day 7, the small intestine tissues and serum of the rats were collected. Hematoxylin-Eosin (HE) staining was used to observe the pathological changes of the small intestinal tissues, and the levels of inflammatory factors, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-10 and lipopolysaccharide (LPS) of small intestine tissues were detected by enzyme-linked immunosorbent assay (ELISA). The levels of diamine oxidase, D-lactic acid and bacterial endotoxin in serum were detected by intestinal barrier function biochemical analysis system. Results: The morphology of hucMSC-EXO was observed to be round or oval, with uniform size and a peak diameter of approximately 100 nm, expressing positive markers CD63 and TSG101. In the sham injury group, the rats' body weight increased by approximately (6.3±1.2) g/day, whereas in the burn group, the body weight significantly decreased on the first day post-injury and then gradually increased at a rate of (1.6±0.5) g daily. In contrast, the body weight of the Burn+EXO100, 200, and 400 groups increased at a rate of (2.9±1.1) g daily. By day 7 post-injury, the body weight in the Burn+EXO200 and Burn+EXO400 groups were significantly higher than those in the burn group and the Burn+EXO100 group (all P<0.05). HE staining showed that the villus height in the small intestine (duodenum, jejunum, ileum) of the burn group [(711±35), (526±25), (418±33) μm] was significantly reduced with severe structural damage, while the small intestine structure and villus height in the EXO-treated groups showed varying degrees of recovery. The villus height in the Burn+EXO200 group [(1 050±40), (798±30), (609±29)μm, respectively] and burn+EXO400 group [(1 102±46), (830±28), (625±33)μm, respectively] recovered significantly (all P<0.05). ELISA results indicated that the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and LPS in the burn group [(29.3±1.7), (81.2±2.5), (582.4±36.9), (22.1±0.6), (366.8±15.9)ng/L, respectively] were elevated, while those were significantly reduced in the EXO-treated groups, with Burn+EXO200 group [(17.9±1.0), (58.2±2.3), (206.6±38.7), (8.9±1.0), (94.9±7.3)ng/L, respectively] showing the most significant reduction (all P<0.05). The level of anti-inflammatory cytokine IL-10 was elevated in the burn group [(293.4±16.0) ng/L], and it was significantly increased in the burn+EXO100, 200, and 400 groups [(591.8±40.7), (672.5±53.7), (712.5±36.2)ng/L, respectively] (all P<0.05). Assessment of intestinal barrier function showed that serum diamine oxidase in the burn group decreased, while D-lactate and bacterial endotoxin increased. The EXO-treated groups demonstrated significant improvement in diamine oxidase, D-lactate, and bacterial endotoxin levels, with Burn+EXO200 group showing the most significant effect (all P<0.05). Conclusions: In this study, hucMSC-EXO was successfully isolated, extracted, and identified. An intraperitoneal injection of 200 μg of hucMSC-EXO demonstrates the most effective repair of intestinal structure and function in rats with severe burn injuries.