AIM: To explore effects of huoxiangzhengqi liquid (HXZQ) on enteric mucosal immune responses in mice with Bacillus dysenteriae and Salmonella typhimurium induced diarrhea (BSD).

METHODS: BSD was induced in Balb/c mice by oral administration with Bacillus dysenteriae and Salmonella typhimurium. HXZQ was administrated from the day of diarrhea induction at dosages of 5.21 g/kg and 0.52 g/kg, respectively. The onset of diarrhea and lasting time were recorded. Peyer’s patches and peripheral lymphocytes were prepared for flow cytometry, and levels of TNF-α in peripheral blood and enteric tissue homogenates were determined with ELISA. Student’s t test was employed for statistics.

RESULTS: Mice in BSD group started showing continuous diarrhea on the day of induction until the fourth day when they were sacrificed. Diarrhea in the mice of HXZQ high and low dose groups lasted for 36 and 54 h, respectively. There were more CD4⁺ and CD8⁺ cells in peripheral blood, fewer CD4⁺ cells in Peyer’s patches in BSD mice compared to normal mice. Fewer CD4⁺ and CD8⁺ cells was shown in the mice in HXZQ high group compared to BSD mice. In Peyer’s patch, there were more CD8⁺ cells in mice in HXZQ high and low dose groups and more CD4⁺ in mice in HXZQ high group. Higher levels of TNF-α in peripheral blood and intestinal tissue homogenates in BSD group were observed. Mice in HXZQ high group showed decreased levels of TNF-α in peripheral blood and enteric tissue homogenates.

CONCLUSION: The immune regulation of CD4⁺ and CD8⁺ cells in Peyer’s patch and suppression of TNF-α levels in enteric homogenates may partially explain the effect of HXZQ on improvement of BSD.

Huoxiangzhengqi liquid (HXZQ), consisting of Agastache rugosa, Ammi majus, areca peel, hoelen, beefsteak plant leaf, aurantii nobilis pericarpium, atractylodes macrocephala, magnolia bark, pinelliae tuber, platycodon root and Glycyrrhiza uralensis, has been used in China for thousands of years for the treatment of various diarrhea[1]. It is one of earliest marketed herbal medicines and is produced by China Tongrentang Pharmaceuticals Corp(GMP certificated), Beijing. Pharmacological research has shown that HXZQ has the activities of improving gastrointestinal dysfunction, anti-inflammation and immune regulation[1-3].

Enteric mucosal responses play an important role in anti-inflammation of the digestive tract[4,5]. However, the effects of HXZQ on enteric mucosal immune responses are largely unknown.

Dysenteriae and Salmonella typhimurium induced diarrhea (BSD) is one of widely used animal models for human infectious diarrhea study[6,7]. In the present study, we investigated the effects of huoxiangzhengqi liquid on enteric mucosal immune responses in mice with BSD.

HXZQ is one of ancient compound liquids of traditional Chinese herbal preparations. In China, it has been used for the effective treatment of various common gastrointestinal infectious diseases (GID), such as acute gastroenteritis, diarrhea and gastrointestinal type Frigorism[1]. It has been reported that HXZQ can promote gastroenteric motility, ameliorate myoelectricity of enteric smooth muscle and regulate gastrointestinal function[2]. Huoxiangzhengqi liquid has also been shown to have regulatory effects on specific immune responses[3]. Agastache rugosa is the essential component of huoxiangzhengqi liquid, and the extract of Agastache rugosa has been reported to have immune modulatory activities[9-15]. BSD in mice is induced by administration of attacking pathogenic bacteria, and shares similar pathogenesis and symptoms with GID[6,7]. Our study showed that HXZQ at 5.21 g/kg and 0.521 g/kg could significantly ameliorate diarrhea in BSD mice.

Although bacteria are usually regarded as harmful, it is now recognized that the breakdown or a failure of components of the indigenous microflora is responsible for allergic diseases or food enteropathies[8-10]. Intestinal microbial species and its product can partially modulate mucosal immune responses and influence the expansion and structure of lymphoid tissues, the activation of TH₁ and TH₂ T cells[11,12]. Intestinal bacteria are required for the development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance[13]. Peyer’s patch lymphocytes, as one important part of GALT, are considered to be the effectors in reacting with intestinal antigens, and mediate immune tolerance[14]. It has been known that some drugs can reestablish the microenvironment of the intestinal tract, thereby enhancing mucosal as well as systemic immune response[15-17]. Intestinal immunity has been evaluated with respect to CD4⁺ cells armed with secretory intestine-derived IgA, and CD8⁺ cells armed with IgG[18]. Down-regulation of immune responses by regulatory T cells is one of the major mechanisms involved in the induction of tolerance and self- and allo-antigen[19]. Microflora-associated antigens can drive antigen-specific CD4⁺ T cells to cause intestinal inflammation. Therefore, the effectors CD4⁺ T cells seem to regulate the development of intestinal inflammation[4]. Our results showed that there were significant changes in CD4⁺ and CD8⁺ cells both in Peyer’s patch and peripheral blood in BSD mice, and HXZQ could regulate the proportion of CD4⁺ to CD8⁺. These results further supported that dysbacteria might take some active role in immune response in BSD mice, and HXZQ might have potential regulative activities on enteric mucosal immune responses in diarrhea. The results also indicate that CD4⁺ and CD8⁺ T cells in Peyer’s patch may serve as one of the targets of the immunoregulatory activity of huxiangzhengqi liquid.

TNF-α is a pleiotropic cytokine with strong proin-flammatory, immunomodulatory properties, and plays a critical role in inflammation and inflammatory bowel disease[20,21]. Anti-TNF therapy has been proven to be a milestone in the treatment of inflammatory bowel disease, and equally important in other inflammation-mediated conditions, including viral infections and mucosal inflammation[22,23]. Different strategies have been explored aiming at inhibiting TNF. Infliximab, a chimeric antibody to TNF-α, holds much promise for the treatment of Crohn’s disease[24]. TNF-α levels in enteric tissue homogenates and peripheral blood in BSD mice were higher in our study, while HXZQ could decrease TNF-α. The results could partially explain the immunosuppressive activities of HXZQ in immune response of mice, and further support that dysbacteria could lead to increased inflammatory response in BSD mice.

In conclusion, enteric immune response might be actively involved in diarrhea and regulation of CD4 and CD8 T cells in Peyer’s patch and suppression of TNF-α levels in enteric homogenates might be one of the mechanisms of HXZQ in the treatment of diarrhea.