Extensive burn injury is a type of severe trauma. As early as 1986, scholars proposed the famous argument that the gastrointestinal tract is the primary organ of multiple organ dysfunction syndrome (MODS)(9). MODS is the root cause of death in severe burn patients(10, 11). Severe burns can induce destruction of the intestinal mucosal barrier and changes in permeability and are closely related to secondary MODS(12). A study showed that intestinal mucosal permeability was significantly increased in mice 1 h after infliction of a 30% TBSA burn, and the morphological analysis of intestinal tissue by HE staining showed that the intestinal barrier was disrupted(13). In this study, healthy and pathogen-free adult male SD rats were selected as the research object. Using a desktop super temperature-controlled scald instrument, burn injury was inflicted by creation of a 4 cm2 scald wound on the dorsal surface at 92 °C for 18 s, establishing a 40% TBSA 3rd degree scald model. The pre-experiment satisfied the skin morphology criteria. The HE staining results proved that the degree of the burns was consistent with severe burns, indicating that the model was successfully established.
Intestinal permeability is one of the most important indicators for evaluating intestinal function(14, 15). In the present study, the burn-induced increase in intestinal permeability to 4.4-kDa FITC-dextran was notably eliminated 6 h after dexmedetomidine treatment, proving the action of repair mechanisms after dexmedetomidine infusion. Therefore, we selected a time point of later than 6 h for subsequent experiments. Intestinal permeability caused by burn injury was accompanied by histological changes in the intestine, decreases in the expression and distribution of the tight junction proteins ZO-1 and Occludin, and increases in inflammatory cytokine levels, in addition to elevation of both MLCK protein expression and MLC phosphorylation. After dexmedetomidine treatment, those burn-induced changes were improved.
The intestinal barrier comprises mechanical, immunological, biological, and chemical barriers(16). The mechanical barrier is a pivotal part of the intestinal barrier and is maintained through intestinal epithelial cells and intercellular junctions(17). Tight junctions consist of multiple proteins such as ZOs, occludins and claudins(18). The intracellular C-terminus of occludin is linked with ZO(19). Those tight junction complexes are vital for maintaining intestinal barrier integrity after injury(20). Reports have indicated that burn-induced intestinal damage leads to reduced expression of tight junction proteins(21), consistent with our results. In addition, we revealed that the expression levels of the classical tight junction regulatory proteins ZO-1 and Occludin were evidently elevated by dexmedetomidine treatment during recovery from burn injury. Their distribution in intestinal tissue was also significantly increased. These findings prove that dexmedetomidine can enhance the expression and distribution of the tight junction proteins ZO-1 and Occludin, thereby ameliorating intestinal barrier damage.
Previous studies have confirmed that inflammatory cytokine-mediated intestinal epithelial barrier dysfunction promotes multiple enteropathies, including IBD(22, 23). These and other inflammatory cytokines disrupt the integrity of tight junction complexes and enhance intestinal barrier permeability. Numerous studies have shown that the levels of some pro-inflammatory cytokines, such as TNF-α, IFN-γ, IL-6 and IL-1β, are notably elevated in mice, rats and human patients with severe burns(24–26). These alterations in pro-inflammatory and anti-inflammatory cytokines may promote burn-induced intestinal barrier injury. In our research, we measured the levels of the pro-inflammatory cytokines TNF-α and IL-6 and the anti-inflammatory cytokine IL-10. TNF-α is an early pro-inflammatory factor that is released mainly by macrophages and monocytes and participates in inflammatory and immune responses. In addition, TNF-α can disrupt the intestinal barrier by decreasing tight junction protein levels and reorganizing the cytoskeletal structure(27). IL-6 is an early inflammatory factor that is rapidly induced in the setting of infections, burns, tumours, etc., and its concentration is increased as a better predictor of disease. IL-10 is a potent cytokine inhibitor of macrophages that moderates inflammation. Our findings indicated that dexmedetomidine can downregulate the expression of the pro-inflammatory factors TNF-α and IL-6 and promote the expression of the anti-inflammatory factor IL-10. These conclusions are consistent with the results found by Chen et al(4). Our outcomes verified that inflammatory cytokines can damage the intestinal epithelial barrier by decreasing tight junction protein levels, while dexmedetomidine treatment can reverse this trend.
Tight junctions are in a state of relatively stable dynamic remodelling and are regulated by various factors, such as mitogen-activated protein kinases (MAPKs), Ca2+-induced E-cadherin, phospholipase C, protein kinase A, Rho GTPase, tyrosine kinase, and MLCK(8). Among these factors, MLCK plays a vital role in the regulation of tight junction dynamics(28). To explore the causes of the structural damage to tight junctions during burn-induced intestinal injury, we considered the MLCK/p-MLC pathway, which has been illustrated to induce disruption of tight junctions after MLC and MLCK phosphorylation. MLC phosphorylation alone has been proven to be adequate to induce tight junction regulation and to enhance tight junction permeability, which is closely related to morphological and biochemical changes in the tight junction proteins ZO-1 and Occludin(29). Our research showed that burn-induced intestinal barrier damage was accompanied by enhancement of MLCK protein expression and MLC phosphorylation. However, dexmedetomidine not only suppressed the burn-induced increases in MLCK expression and MLC phosphorylation but also reduced intestinal barrier permeability, ameliorated intestinal pathological damage, and increased the expression and distribution of the tight junction proteins ZO-1 and Occludin. Therefore, these experiments demonstrated that the MLCK-dependent MLC phosphorylation signalling pathway may be related to burn-induced intestinal barrier injury.