The small intestine is made up of labile cells which are particularly sensitive to IR damage, and intestinal IR injury is a common event under various clinical conditions. Intestinal IR can cause epithelial cell damage, disrupt mucosal integrity and small intestinal function, and subsequently increase mucosal and vascular permeability, systemic inflammation, multiple organ dysfunction, and even death. There is currently no effective treatment for intestinal IR injury. In this study, we investigated the protection of Qingchang mixture on the integrity and function of the small intestinal mucosa using the rat intestinal IR model and IEC-6 cells HR model in vitro, providing an effective clinical treatment for intestinal IR injury.
The HE staining results showed obvious intestinal histopathological damage in the rat intestinal IR model. The changed expression of MDA, SOD, and GSH-Px indicated the oxidative stress state of the rat intestinal. The secretion of pro-inflammatory factors (IL-6, IL-1β and TNF-α) suggested the occurrence of local inflammation. The expression levels of ALT and AST indicated the damage to distant organs. Down-regulation of TJ proteins (such as ZO-1) further supported the TJ destruction and intestinal barrier dysfunction. TJ consists of a group of transmembrane proteins, including occludin, claudins, and ZO-1, which acts as a closed barrier by maintaining cell polarity and internal environment balance and regulating the permeability of the epithelial cell space24. IR injury results in the destruction of TJ, which is characterized by altered and redistributed TJ protein expression25.
Importantly, our research found that Qingchang mixture effectively prevented the pathological damage and permeability changes caused by IR. Compared with the IR group rat model, the administration of Qingchang mixture significantly reduced the Chiu score, the expression of MDA, and the secretion of pro-inflammatory factors, ALT and AST, and significantly increased the expression of SOD, GSH-Px and TJ proteins. This is the first report that Qingchang mixture can prevent intestinal pathological damage and barrier dysfunction caused by IR. In addition, studies have shown that changes in TJ protein expression are partially mediated by TNF-α26. One research suggests that Qingchang mixture can significantly promote the intestinal motility of mice, has no obvious stimulating effect on gastric mucosa. The conventional dosage is safe, and the lethal dose of the acute toxicity experiment mouse was 13.95 g/kg27. Regarding the dosage and mode of Qingchang mixture, more research is needed. In addition to the rat intestinal IR model, we also used the IEC-6 cell HR model in vitro to investigate the protective effect of Qingchang mixture on the intestinal epithelial cells. The pretreatment of Qingchang mixture improved the viability, and hindered apoptosis of the HR-induced IEC-6 cells.
The intestinal epithelium is constantly exposed to various bacteria and bacterial products. The biological response to endotoxin is mediated by the TLR4 complex, which leads to the activation of NF-κB and the release of cytokines, including IL-6, IL-1β and TNF-α28, 29. One research reveals that the expression of TLR4 and NF-κB is increase after intestinal IR in mice12. One report shows that TLR4 has a protective effect on intestinal IR damage using TLR4 knockout mice13. Chen et al. also suggest that administration of the representative TLR4 ligand LPS, reduces the incidence of intestinal IR damage30.
Contrary to these studies, some reports indicate that the TLR4 signaling pathway is an exacerbating factor for intestinal IR damage. Pope et al. demonstrate that TLR4 deficiency reduces intestinal IR damage by reducing complement activation14. Zhu et al. find that the TLR4 mutant significantly reduced intestinal IR damage partially by reducing inflammatory response and oxidative stress15. Moses et al. believe that TLR4-mediated COX-2 expression exacerbates intestinal IR damage16. In addition, the interaction of TLR4 and endogenous TLR4 ligand high mobility group protein 1 (HMGB1) increases mouse intestinal IR damage17. Other studies have revealed the role of the TLR4/NF-κB signaling pathway in non-human primates intestinal IR damage. Wu et al. point out that macaques develop multiple organ dysfunction after intestinal IR injury, which is related to the expansion of the severe innate immune response31, 32. This response is mediated by the general activation of the TLR/NF-κB/cytokine pathway throughout the small intestinal mucosal system.
Due to the wide variety of TLR4 ligands, the role of the TLR4 signaling pathway may vary depending on the activation ligand. In addition, unlike other organs, the gut has always coexisted with bacteria. The identification of intestinal bacteria by TLRs plays an important role in controlling intestinal homeostasis and preventing damage22. Therefore, the balance between the beneficial and deleterious effects of the TLR pathway may be disturbed by intestinal flora colonies33.
In this study, we found the activation of TLR4/NF-κB pathway in a rat intestinal IR injury model. The administration of Qingchang mixture inhibited the activation of TLR4/NF-κB pathway. In addition, treatment with TLR4 or NF-κB specific inhibitors effectively protected intestinal function. In other words, Qingchang mixture was likely to protect the intestinal function by inhibiting the activation of the TLR4/NF-κB pathway.