The Underlying Mechanism Of Berberine In Diarrhea Disease

Background:Diarrhea is one of the most common and important diseases, which has a major impact on morbidity and mortality worldwide. About four billion cases occur each year and as many as 3～4 million individuals are claimed to death annually. Although many therapy strategies and drugs have been adopted, diarrhea remains a serious disease worldwide. Several million people died from diarrhea every year, especially in developing countries. Under physiological condition, intestinal epithelial cells are equipped with a number of ion channels, carriers, and pumps, located either on the luminal or basolateral membrane, making absorption process and secretory process under well-balanced. However, when a serial of diarrhea stimulations exert effects on intestinal mucosa, including enteric bacterial infections, hormonal factors etc, the expressions or activities of ion channels, carriers, and pumps are altered, thus the balance between absorption and secretion is disturbed which can make excessive fluid and electrolyte loss and even cause patients to die. During the past two decades there has been a continuing search for drugs that might affect the secretory or absorption process, and a number of candidate drugs have emerged. As one of the candidate drugs, berberine has been proved to be an effect drug for diarrhea. As a traditional Chinese medicine, berberine has been used to treat diarrhea for a long history. Many studies have demonstrated its significant antimicrobial activity against a variety of organisms including bacteria, viruses, helminths, and Chlamydia, but antimicrobial activity itself can't fully account for its anti-diarrhea effect, especially in secretory diarrhea. In animal model, berberine could reverse the secretory diarrhea induced by cholera toxin, which implied that berberine might exhibit its anti-diarrhea effect partially by changing ion transport; likewise, randomized controlled trial indicated that berberine was an effective and safe anti-secretory drug for diarrhea caused by enterotoxigenic Escherichia coli and Vibrio cholerae, suggesting the underlying mechanisms still need to be explored.Aims:1. To establish diarrhea mice model and observe the anti- diarrhea effect of berberine. 2. To explore the effect of berberine on the expression of ion channels or carriers. 3. To explore the molecular mechanisms by which berberine might affect the expression of ion channels or carriers.Methods:1. Male BALB/c mice (20～22 g) were divided into 3 groups at random. In control group, mice were repeatedly given 0.3ml normal saline ; in diarrhea group, mice were repeatedly given 20mg/kg sennosideA with intragastric feeding needles twice per day, and those with profuse liquid stool were recorded as diarrhea-positive animals; in treatment group, 30min after given sennosideA, each mouse received 80mg/kg berberine for treatment. Each mouse was fed in single cage rebased with white filter paper. The frequency of liquid stool within 12 h was counted, and the change of body weight was recorded. 2. Epithelial cells were scraped from mice proximal colon and total RNA was extracted. Semi RT-PCR was adopted to detect the expression of ion channels and carriers which affect the electrolyte transport in human intestine. Furthermore, immunohistochemistry was adopted to confirm the result of RT-PCR. 3. Human intestinal epithelium cell line (HIEC) was treated with sennosideA or co-treated with sennosideA and berberine. The expression of ion channels and carriers were detected by Semi RT-PCR and western blot analysis.Results:1.In control group, no mice appeared diarrhea; In diarrhea group, all mice developed intense diarrhea 1～1.5h after 20mg/kg sennosideA was administrated, and the diarrhea could persist for 5～6h. However, in berberine treated group, mice rarely appeared fluid stool. The frequency of liquid stool among control group, diarrhea group and treatment group was significantly different (0±0.00 VS 8.57±0.1 VS 0.14±0.05, p<0.05). 2. The mRNA expressions of ion channels and carriers were determined by semi RT-PCR in animal model. Compared with the mice treated with berberine, the expression of NHE3 and AQP4 were significantly decreased in proximal colons of the diarrhea mice. Similarly, in HIEC co-treated with sennosideA and berberine, the mRNA expression of NHE3 was 3 times as high as that in HIEC only treated with sennosideA, and for AQP4 it was 2.5 times. 3. In mice model, the proximal colon was chosen to perform IHC analysis. Both NHE3 and AQP4 were localized on the epithelia surface of the proximal colon. In diarrhea mice, colonic mucosal epithelial cells displayed very weak expression of NHE3 protein; however, the NHE3 protein was highly expressed in mice treated with berberine. Likewise, slight AQP4 expression was observed in the brush border membrane of proximal colon of the diarrhea mice, but the expression was significantly enhanced after exposed to berberine. 4. In order to confirm the result that was indicated by IHC analysis, Western blot analysis was performed in HIEC cell lines. NHE3 protein expression was decreased by approximately 50% in HIEC treated with sennosideA compared with that co-treated with sennosideA and berberine. Similarly, AQP4 protein expression was increased by two-fold after exposed to berberine.Conclusions:1. SennosideA is an effective material to induce diarrhea, and berberine is an effective drug for the diarrhea which is induced by sennosideA. 2. Berberine can increase the expression of NHE3 and AQP4 in diarrhea mice, suggesting berberine might exhibit its anti-diarrhea effect partially by enhancing the absorption of Na~+ and water. 3. Similarly, berberine affects the expression of NHE3 and AQP4 in HIEC cell line.