Psoralen, the main active component of psoralea, has many effects, such as cartilage protection and anti-tumour, immune regulation, and anti-vitiligo activities[12]. Recent studies have also found that psoralen can stimulate osteoblast differentiation by activating BMP (BMP, bone morphogenetic protein) signals[13]. Psoralen can inhibit bone resorption of osteoclasts in vitro[14]. Additionally, psoralen can treat arthritis by regulating the balance of Th1/ Th2 cells and inhibiting the expression of TNF-α, IL-6, and IL-1β[8]. Furthermore, it can also increase the bone mass of ovariectomised rats with osteoporosis by stimulating bone marrow mesenchymal stem cells to differentiate into osteoblasts[9, 12].
Chronic periodontitis is a chronic infectious disease, characterised by gingival inflammation, periodontal pocket formation, and alveolar bone resorption. Porphyromonas gingivalis is the main dominant bacteria in periodontitis, especially in chronic periodontitis. Recently, it has been found that oral administration of Pseudomonas gingivalis caused changes in the composition of intestinal flora, regulated the intestinal immune system, and impaired intestinal barrier function, thereby resulting in systemic inflammation and related pathological changes[15-17]. Among them, the intestinal barrier, as an important factor affecting systemic inflammation, is composed of a mechanical barrier that composed of epithelium and tight junction proteins, an immune barrier composed of immune cells and immune proteins in the intestinal mucosa, and an ecological barrier composed of normal intestinal flora. The three are closely related in function, and together provide intestinal immunity. The function of the intestinal mechanical barrier mainly depends on the transcellular pathway and the paracellular pathway. The basic structure of the paracellular pathway is the tight junction (TJ) composed of the proteins claudin, occludin, and Zo. The occludin and claudin family of proteins together form the transport pathway, which controls epithelial barrier permeability. Epithelial permeability increases when these proteins are overexpressed[18], causing pathogenic bacteria and related metabolites to invade the body. In this study, no significant difference was found in the protein expression of occludin and claudin-5 among the groups, indicating that psoralen had no significant effect on these proteins in the intestinal mechanical barrier.
In the physiological state, the host recognises endogenous or exogenous microorganisms through intestinal NODs, and maintains the gastrointestinal mucosal epithelial barrier by regulating the release of antimicrobial substances and maintaining the homeostasis of intestinal flora. NODs interact with symbiotic bacteria, and symbiotic bacteria promote the expression of NODs. Simultaneously, NOD negative feedback inhibits the proliferation of symbiotic bacteria. NODs play an important role in maintaining the balance between intestinal symbiotic flora and host immune response[19-20]. NOD2 is an innate immune protein in gastrointestinal epithelial cells[21], which can recognise the teichoic acid dipeptide in the bacterial wall and mediate the activation of NF-κB (NF-κB, nuclear factor kappa-B) and the expression of the inflammatory cytokine TNF-α[20, 22], thus initiating the immune response to pathogens[23]. In this study, the expression of NOD2 in the intestinal mucosa of the Model group was found to be higher than that of the Control group, indicating that the intestinal immune barrier was activated. After intragastric administration of psoralen, the expression of NOD2 protein in the Peri+Pso group was significantly lower than that of Model. Psoralen alleviated intestinal injury and decreased the expression of NOD2 to some extent.
Recent studies have shown that bone metabolism such as bone growth and remodeling is closely related to intestinal microflora[24-25]. Intestinal flora can maintain homeostasis of the host immune system, regulate the activity of osteoclasts and osteoblasts, and affect the balance of bone metabolism[26]. In this study, analysis of the alpha index of intestinal flora showed that the Chao1 index of the rats in the Pso group was significantly higher than that in the Control group, and the Shannon index in the the rats in the Peri+Pso group was significantly higher than that in the Model group. The results showed that psoralen increased the abundance and diversity of intestinal flora in rats. Atarashi et al. [27] found that Clostridium can promote the number and function of Treg cells, form an environment rich in TGF-β (TGF-β, transforming growth factor-β), and inhibit the formation of osteoclasts. In addition, some studies have found that the Clostridium leptum subgroup can promote the production of Treg cells and secrete anti-inflammatory cytokines (such as IL-10) to inhibit self-traumatic inflammation. In this study, LEfSe analysis of intestinal flora in rats also found that the relative contents of o_Clostridiales and c_Clostridia in the Pso group increased, indicating that psoralen can increase the relative content of Clostridium in the intestines of healthy rats.
In addition, the F/B ratio was considered to be another significant indicator of microbial structure, as determined by the taxonomic composition[28]. In some studies on human osteoporosis, it was found that the F/B ratio in the osteoporosis group was higher than that in the control group[29-30]. However, we found that the decrease of the number of Firmicutes and the increase of the number of Bacteroidetes in the Pso and Peri+Pso groups led to the decrease in the F/B ratio and the decrease in ABL in the Peri+Pso group. It is suggested that psoralen can reduce the F/B ratio and ABL in periodontitis rats. At the same time, LEfSe analysis of intestinal flora showed that the relative content of p_Bacteroidetes in the Peri+Pso group increased. However, studies on senile osteoporotic rats and humans with low bone mineral density also found a decrease in the F/B ratio[31-32]. This was similar to the increase in bone loss and the decrease in the F/B ratio in the Model group compared with the Control group in this study. Therefore, the role of the F/B ratio as a significant indicator of bone mineral density needs to be further elucidated.
The aforementioned results indicate that psoralen had a certain effect on the intestinal flora and immune barrier, to verify whether psoralen affected the intestinal tract and alveolar bone resorption. We further statistically analysed M2 ABL in rats, and found significant ABL compared between the Control, Model, and Peri+Pso groups; however, ABL in the Peri+Pso groups was significantly less than that in the Model group. It was suggested that psoralen can reduce alveolar bone resorption in periodontitis rats, and no significant difference was found in ABL between the Control and Pso groups. Combined with the results of NOD2 protein expression analysis in the ileal mucosal immune barrier in rats, psoralen had a significant effect during periodontitis, however, no significant change was observed under normal conditions. We speculate that psoralen mainly plays a role in inflammation and is related to the immune response.
Heredity, hormones, immune system function, nutrition, and lifestyle factors play a decisive role in bone tissue mineralisation. Several mechanisms accelerate bone loss by altering the activity of osteoblasts and osteoclasts. For instance, bone mass loss mediated by oestrogen deficiency is associated with osteoclast differentiation and activity, in part due to the increased production of pro-inflammatory cytokines (TNF- α, IL-1, and IL-6) and the activation of immune cells[33]. At present, some scholars have proposed that intestinal bacteria can lead to the activation of T cells in bone tissue. Inflammatory cytokines IL-6 and TNF-α secreted by T cells can promote gingival inflammation, accelerate periodontal connective tissue destruction, and alveolar bone resorption. TNF-α can induce the production of RANKL and macrophage colony stimulating factor (M-CSF) and downregulate OPG[34-35]. TNF-α can also directly interact with osteoclast precursors and promote their differentiation and maturation. Through the receptor P55, TNF-α can enhance the response of bone marrow macrophages (BMM) to M-CSF and RANKL, and indirectly promote BMM to differentiate into osteoclasts. However, IL-10 can inhibit T lymphocyte synthesis and production of IL-6, TNF-α, and interferons[36-38]. Therefore, we further detected the expression level of RANKL and OPG in periodontal tissues, and the levels of inflammatory cytokines IL-6, IL-10, and TNF-α in serum. This study found that the expression of OPG increased and the expression of RANKL decreased after administration of psoralen. The RANKL/OPG ratio in the Model group was higher than that in the Control group, and decreased after treatment with psoralen. At the same time, we found that there was no significant difference in IL-6 expression between the four groups, however, the content of TNF-α in the Peri+Pso group was significantly lower than that in the Model group. Additionally, the anti-inflammatory factor IL-10 in the Peri+Pso group was significantly higher than that in the Model group. Therefore, psoralen can promote the secretion of IL-10, inhibit the secretion of TNF-α, and slow down the inflammatory reaction. In conclusion, psoralen may affect the intestinal immune barrier and ecological barrier, mediate immune response, promote the secretion of the anti-inflammatory factor IL-10, and reduce the secretion of pro-inflammatory factor TNF-α, thus reduce the expression of RANKL and reduce ABL in experimental periodontitis in rats. These results suggest that psoralen can potentially be used in the treatment of periodontitis.