Female Sprauge-Dawley rats weighing 200-250 g were used in the study. The animals were fasted for 24 h before the experiment and allowed food and water ad libitum after induction of colitis. The study was approved by the Ethic Committee of Wuhan University Medical School.

Colitis was induced by a method of hapten-induced colonic inflammation as previously described[24]. Rats were anaesthetized by intraperitoneal administration of 100 g/L urethane. A small volume of 2, 4, 6-TNBS (Sigma Company) was dissolved in 50% ethanol to a final concentration of 100 mg/mL, and 0.3 mL of TNBS solution was intracolonically administrated with a polypropylene catheter by inserting 8 cm via the anal canal. Rats in a group were given 150 U/kg LMWH (dalteparin made by Pharmacia & Upjohn Company) subcutaneously 1 h before induction of colitis, and went on once a day for 6 d. Then a half dose of LMWH was given for the next 7 d. Treatment of the LMWH 300 U/kg group was the same as the 150 U/kg group, but the dose of LMWH was as high as 300 U/kg. Control animals received the same volume of normal saline once a day instead of LMWH after treated by TNBS.

Animals in each group were sacrificed at 24 h, d 7 and 14 after induction of colitis. The colon was isolated and a segment of colon was excised for the evaluation of macroscopic and histological findings and also for TNF-α immunohistochemical assay. A blood sample was drawn from heart of the rats for determination of platelet surface P-selectin and serum IL-8 before the rats were sacrificed.

Macroscopic evaluation of colonic damage was conducted by two authors (HH, LLG) and mucosal hyperemia, ulcers, inflammatory exudation and bleeding were recorded. The most damaged site of the colon was chosen as the part for histological studies. For control group the colon at 8 cm above anus was excised for histological studies.

For histological examination, formalin-fixed tissues were embedded in paraffin and 5 μm -thick sections were stained with hematoxylin and eosin, and evaluated under light microscope by a pathologist (GSG) blinded to the experimental protocol. Colonic damage was assessed by the grades described by Fedorak et al[25]. Mucosal ulceration: 0: no-ulceration; 1: focal ulceration; 2: multifocal-ulceration; 3: diffuse ulceration. Depth of injury was graded as follows: 0: no injury; 1: mucosal involvement only; 2: mucosal and submucosal involvement; 3: transmural involvement. The ulceration and depth of injury grades were scored and put together as a result ranging between the minimum of 0 and maximum of 6.

Platelet surface P-selectin molecules were detected by radioimmunoassay. The kit was purchased from the Institute of Thrombosis and Homeostasis of Suzhou University, China. Briefly, 2 mL of anti-coagulated blood was taken from the heart of rats and platelets were counted under microscope. The blood was fixed by 2 g/L glutaraldehyde in PBS solution at room temperature for 30 min and stored at 4 °C for determination of platelet surface P-selectin. The fixed blood with 2.5 × 10⁶ platelets was divided into 3 tubes and washed with 1 mL of 0.01 mol/L PBS, pH7.4, per tube and centrifuged at 2500 rpm for 10 min. Supernatants were removed. 50 μL of SZ-51mab labeled with ¹²⁵I was added into the reaction tubes and mixed fully. 15 μL of SZ-51mab without ¹²⁵I was added into control tubes. Each tube was incubated at 37 °C, washed 3 times with PBS, and centrifuged at 3000 r/min for 10 min. Cpm of precipitations in each tube was measured by a γ reader.

Serum IL-8 production was assayed by ELISA sandwich method. The IL-8 ELISA kit was purchased from the Institute of Immunology of the Fourth Military Medical University, China. Briefly, 96-well plates were precoated with monoclonal antibody specific to human IL-8. Serum samples, negative control and diluted IL-8 standard markers were added into the plates. The serum samples were detected according to the procedures described in the protocol. A₄₁₀ value was read by the ELISA reader.

Expression of TNF-α in colonic mucosa was assessed by immunohistochemistry. The regents of TNF-α were purchased from Beijing Zhongshan Biotechnology, China. Positive expression of TNF-α was brown deposited granules in the plasma of neutrophils, monocytes and lymphocytes. The grades of expression of TNF-α in mucosa were classified as follows: 0: no staining; 1: a few maple granules, or a few fine brown granules, not exceeding the 1/4 total area of cytoplasm; 2: uniformity maple in the whole cytoplasm or wide brown granules in the cytoplasm, not exceeding the 1/2 area of cytoplasm; 3: the cytoplasm was full of brown granules with a lower density; 4: the total cytoplasm was full of crassitude dark brown granules, and covered whole nuclei of the cell. One hundred cells were counted under oil microscope, positive cells were recorded and positive cell rate was calculated. The scores of expression of TNF-α were calculated by sum of each grade multiplying its positive cell rate.

Data were expressed as mean ± SD. Data in different groups were analyzed by ANOVA and post multiple tests (Tukey-Kramer or Student-Newman-Keuls). A P value less than 0.05 was considered statistically significant.

Control animals subjected to intracolonic administration of TNBS in 500 ml/l ethanol showed colonic mucosal injury with ulceration. Signs of the mucosal damage were monitored for 14 d. As early as 24 h after TNBS treatment, colonic mucosa was shown to have hyperemia, congestion, erosion, hemorrhagic ulcerations in the injured site. The damage was still maintained at d 7 and appeared to be multiple ulcerations and partial epithelial necrosis. On d 14, the colonic ulceration still existed. In both doses of LMWH treated groups, mucosal hyperemia, congestion and ulcerations in the injured site were slighter than those in control group. However, mucosal hemorrhage in both LMWH treated groups was severer than that in control group at 24 h, but was resolved on d 7 and 14, respectively.

In control group a large number of neutrophils, monocytes and eosinophils infiltrated in mucosa and submucosa at 24 h and the damage reached peak on d 7. As shown in Table 1, the histological grades according to Fedorak et al[25] were greatly higher on d 7 compared with those at 24 h (P < 0.01). On d 14, the damage of colon was mild, but still had ulceration, chronic inflammatory cell infiltration, vesculitis and granulation tissue formation. In contrast, in both 150 U/kg and 300 U/kg LMWH treated groups colonic damage was mild. Table 1 shows that the histological injury grades in the 300 U/kg LMWH group were decreased at 24 h and on d 7 after treatment, and much obviously on day 14 compared with the control group (P < 0.01).

As shown in Table 2, the scores of expression of TNF-α in mucosa were greatly higher in the first 24 h and decreased on d 7 and 14 after induction of colitis, but there were no significant differences among the three groups except a difference at 24 h between 300 U/kg LMWH treatment group and 150 U/kg LMWH treatment group.

Expression of platelet surface P-selectin was increased on d 7 and 14 in all three groups as shown in Table 3, but reached a significant level only in control group. There were no significant differences among these three groups at the three time points, 24 h, d 7 and 14. Production of serum interleukin 8 was higher at 24 h in the three groups, but significantly decreased on day 14 compared with that at 24 h in 300 U/kg LMWH treated group as shown in Table 4.