Effect Of Balsalzine On Intestinal Mucosal Permeability Of Ulcerative Colitis And Its Mechanism

Background Intestinal mucosal barrier plays a pivotal role in preventing microorganism and toxinum from invading into organism. It is impaired obviously during inflammatory bowel disease (IBD). As a result, a large quantity of endotoxin invades into systemic circulation through the impaired intestinal mucosa, while inflammation and immunologic reaction of the intestine mucosa is induced or aggravated. Therefore, restoring the impaired intestinal mucosa is beneficial for controlling or relieving the inflammation and immunologic reaction in the intestinal mucosa. 5-ASA is used to treat UC for its obvious effect on controlling or relieving the inflammation. Balsalzine (5-ASA azo bonded to an inert carrier, 4-amino-benzoyl-alanine), as a new preparation, which also can release 5-ASA by the cleavage of intestinal luminal bacterial azo reductase, is efficacious for the induction of remission in mild to moderate UC. However, whether it could ameliorate intestinal mucosal permeability is still unknown. Therefore, to study the effect of Balsalzine on intestinal mucosal permeability of experimental colitis and the possible mechanism is important to the studies of UC drugs and its mechanism. .Objective To establish the C57BL/6J mice model of UC induced by DSS and investigate the effect of balsalazine on intestinal mucosal permeability of experimental colitis and its possible mechanism.Methods (1) To establish the mice model of UC: 45 C57BL/6J mice were allowed to drink the distilled water containing 5% (wt/vol) DSS freely for 7 days. In order to verify the model, the disease activity index (DAI) score was evaluated every day; colon tissue was collected for assessment of histological changes by HE, and homogenated for assessment of MPO activity at the end of the experiment. (2) To investigate the effect of balsalazine on intestinal mucosal permeability of experimental colitis and its possible mechanisms: 45 C57BL/6J mice were divided randomly into five groups: normal group, DSS group, three balsalazine groups at different doses. Mice in the normal group was only fed with distilled water freely while in the DSS group and balsalzine groups were fed with 5% DSS for 7 days. Balsalzine was given at doses of 42, 141, 423 mg/kg by intragastric administration once a day from the d1 day of the experiment lasting for 7 days, while mice in the nomal group and DSS group were given the distilled water by intragastric administration as contrast. DAI was evaluated daily. After 7 days, the end of the experiment, the colon tissues were collected for the assessment of histological changes, the content of MDA and the activities of MPO, SOD and GSH-PX. The small intestinal mucosa was collected for the assessment of the content of tumor necrosis factor (TNF)-αand interferon (IFN)-γ, the detection of transmission electron microscope (TEM) and the permeability by Evans blue, the expression of E-cadherin and VCAM-1 by IHC and the small intestine sacs in vitro.The model of Caco-2 damaged by DSS was established and the effect of balsalazine on the model was studied.Results (1) In the normal group, the mice showed normal stool and no obvious weight loss. The the DAI score and histological index (HI) score were not high. The activity of MPO in colonic homogenate was lower than that in the DSS group and balsalazine groups. Compared with normal group, the mice in the DSS group all manifested severe weight loss associated with hematochezia and diarrhea. The DAI and HI score increased significantly (P<0.01), and the activity of MPO of colon also increased. However, in balsalazine groups, the general condition of mice was obviously improved, the DAI and HI score decreased significantly (P<0.01) and the activity of MPO of colon also decreased. (2) Compared with the normal group, the activities of SOD and GSH-PX decreased significantly in the DSS group, while the content of MDA and the levels of TNF-αand IFN-γincreased significantly (P<0.05 ). The intestinal mucosa showed a focal reduction in thinning of microvillous carpet, and even a total disarrangement of epithelial surface, decurtated and broaden junctional complex and enlarged intercellular space under TEM observations. The expression of E-cadherin decreased but the reverse result of the expression of VCAM-1. The amount of Evans blue permeated into the intestinal wall was obvious. Compared with DSS group, balsalzine was found to increase the activities of SOD and GSH-PX and decrease the content of MDA and the levels of TNF-αand IFN-γin colitis mice(P<0.05 ). At the same time, balsalazine was demonstrated to ameliorate microvillus and tight junctions, increase the expression of E-cadherin but decrease the expression of VCAM-1, and decrease the amount of Evans blue permeating into the intestinal wall. Caco-2 cells were damaged obviously by DSS and the damaging effect could be obviously attenuated by balsalazine in different concentrations. It indicated that balsalzine played a role in protecting enterocytes.Conclusion (1) The mice model of ulcerative colitis could be successfully induced and readily applied in various studies of ulcerative colitis such as the studies of the mechanisms and the drugs. (2) Basalazine could decrease DAI and HI score in the colitis model and showed the obvious pharmacological effect of anti-colitis in the present study. (3) The intestinal mucosal permeability was increased in colitis mice, both the intestinal function and microstructures were damaged obviously. Balsalazine could significantly ameliorate the intestinal mucosal permeability. (4) The mechanisms of balsalazine restoring the impaired intestinal mucosa might partially associate with its antioxidative and anti-inflammatory effect. Background Intestinal mucosal barrier is one of the most important barriers of our bodies. It can prevent pathogenic microorganism and toxinum from invading into organism. However, it is impaired during inflammatory bowel disease (IBD). As a result, microorganism and toxinum might invade into systemic circulation through the impaired intestinal mucosa, while inflammation and immunologic reaction of the intestinal mucosa is induced or aggravated, enterogenic infection even multiple system organ failure might also be induced. Restoring the impaired intestinal mucosa is beneficial for controlling or relieving the inflammation and immunologic reaction in the intestinal mucosa and it can prevent severe complications. It is one of the most important methods used to treat IBD. Therefore, it is quite important to detect the intestinal mucosal permeability early. Macromolecules marked by radionuclide are usually used to detect the intestinal mucosal permeability of UC patients by foreign researchers. Observation of the structures of epithelium by intestinal mucosa biopsy is also used to estimate the intestinal mucosal permeability. However, these methods may cause a new damage to patients, or show disadvantages in the sensitivity and specificity to diagnosis. Measurement of urinary lactulose and mannitol by high performance liquid chromatography with a refractive index detector (HPLC-RI) after subjects took orally the lactulose and mannitol liquid is a atraumatic method for estimating the intestinal mucosal permeability. It also has a higher sensitivity and specifity and has been widely used in clinical practice. However, it has not been reported that the measurement of urinary sugars by HPLC-RI for estimating the intestinal mucosal permeability of UC patients.In addition, the intestinal mucosal permeability may be changed before the intestinal inflammation and when the intestinal inflammation is aggravated, it was increased. The intestinal mucosal permeability correlates with the states of diseases and protecting the intestinal mucosal barrier early is beneficial for controlling the condition and increasing the therapeutic effects. Therefore, to detect the intestinal mucosal permeability of UC patients is beneficial for predicting the disease activity of UC patients and increasing the therapeutic effects. In this study, we studied the intestinal mucosal permeability of UC patients and the correlation between intestinal mucosal permeability and clinical indexes which is in order to investigate the sensitivity of the intestinal mucosal permeability to reflect the disease activity of UC patients.Objective To establish the method of HPLC-RI to detect the intestinal mucosal permeability of UC patients and study the correlation between the intestinal mucosal permeability , ESR ,CRP and Sutherland DAI in order to investigate the sensitivity of the intestinal mucosal permeability to reflect the disease activity of UC patients.Methods (1) Establish the method of HPLC-RI to detect lactulose and mannitol in urine. The samples of urine dealed with high speed centrifugation and ion exchange treatment were detected with a refractive index detector and a Hypersil NH2 collumn(4.6 mm×250mm, 5um). The mobile phase was degassed acetonitrile in distilled water (80/20 by vol). (2) Collect all the urine of 15 UC patients and 15 healthy people in 6 hours after they took orally the lactulose and mannitol test solution which was 50ml. Detect the lactulose and mannitol in urine and calculate the ratio of them. Compare the intestinal mucosal permeability between the UC patients and the healthy people. (3) Analyze the correlation between the intestinal mucosal permeability, ESR, CRP and Sutherland DAI in order to compare the L/M, ESR and CRP to reflect the disease activity of UC patients.Results (1) Lactulose and mannitol in urine could be well separated by the method of HPLC. The recovery of lactulose in 3 different concentrations (high, middle and low) was 94.88%, 89.68% and 86.91% respectively. The recovery of mannitol in 3 different concentrations (high, middle and low) was 94.39%, 88.92% and 83.39% respectively. The test results of linearity of lactulose and mannitol were good at the concentrations of 0.05 -5g/L. The CVs of lactulose and mannitol at concentrations of 5g/L, 1.25g/L, 0.15625g/L were all lower than 15%(n=5). By this method, the minimum detectable concentrations of lactulose and mannitol were both 0.05g/L. (2)The lactulose/mannitol ratio in the 15 UC patients was 0.494±0.190, it was significantly higher than that in the 15 healthy people (0.039±0.014, P<0.05). The result indicated that the intestinal mucosal permeability of UC patients was significantly higher than that of the healthy people. (3) There was a strong correlation between the intestinal mucosal permeability and Sutherland DAI of UC patients. The ESR, CRP were normal in the UC patients with the lesion of the left side colon, while ESR, CRP were increased in the UC patients with the lesion of the whole colon (P<0.05).Conclusion (1) The linear correlation of this method of HPLC-RI to detect the lactulose and mannitol in urine is good. The specificity, the precision, the absolute recovery and the sensitivity of it were all high. (2)The intestinal mucosal permeability of the UC patients increased significantly than that of the healthy people. The lactulose/mannitol ratio could reflect the intestinal mucosal permeability of UC patients. (3)The intestinal mucosal permeability could reflect the disease activity of UC patients and the sensitivity is higher than that of ESR and CRP.