Abstract: Objective:To investigate the expressions of T helper cell 1 (Th1)associated chemokine receptors CXCR3, CCR5 and T helper cell 2 (Th2)associated chemokine receptor CCR3 in the spleen tissues of rats with cirrhosis and hypersplenism and probe into the balance between Th1/Th2 lymphocyte subsets.
Methods Experimental study was adopted. Fortysix male SD rats were randomized into the hypersplenic group (n=36) and the control group (n=10). In the hypersplenic group, the rats were fed with 40% CCl4 peanut oil solution (3.0 mL/kg, twice per week) and 15% white spirit for 8 weeks to build the hypersplenic model. The rats in the control group received normal feeding. The animal models with cirrhosis and hypersplenism were confirmed by liver function test, routine blood test, HE staining and Masson staining after visual inspection. The expressions of chemokine receptors of CXCR3, CCR5 and CCR3 were detected by immunohistochemical staining and Western blot. Measurement data with normal distribution were presented as ±s. Comparison between groups was done using the independent sample t test.
Results:Results of visual inspection: the rats in the hypersplenic group suffered from severe hairshedding, metal fatigue and inappetence, with hair dimming and inactivity. There were rats dead successively 5 weeks after model establishment and 19 rats finally survived. The rats in the control group had color and gloss hair, with good appetite and spirits. They were active and sensitive to external stimulation. Changes of pathological morphology in liver: in the hypersplenic group, the fibers became denser and disordered, making normal structure of liver tissues destroyed. The hepatic lobules separated by fibrous bundle and proliferative hepatic cell mass were segmented and surrounded by thick fibrous,leading to the formation of pseudolobule.Disorganized hepatocytes suffused adipocytes, the nucleus of heterocysts enlarged or even multinucleated cells appeared. There was no change in the control group. Changes of pathological morphology in spleen: the rats in the hypersplenic group had slightly swelling spleen with the areas of red pulp increased and whit pulp disappeared gradually. Vascular endothelium became thicker and proliferated. Thickened central artery and fibrosis were depicted. Splenic sinusoid extended. There was no change of spleen tissues in the control group. Changes of liver function: the levels of ALT and AST of rats were (264±111)U/L and (687±299)U/L in the hypersplenic group, which were significantly different from (27±8)U/L and (124±20)U/L in the control group (t=5.64, 4.98, P<0.05). The level of total protein was (54±8)g/L in the hypersplenic group, which was significantly different from (65±3)g/L in the control group (t=-3.35, P< 0.05). Changes of peripheral blood cell count: the white blood cell (WBC) count in the hypersplenic group was (23.9±5.0)×109/L, which was significantly different from (6.2±2.4)×109/L in the control group (t=3.50, P<0.05). The red blood cell count and platelet count in the hypersplenic group were (6.3±0.7)×10 12/L and (418±124)×109/L, which were significantly different from (8.0±0.6)×10 12/L and (1 109±161)×109/L in the control group (t=-2.28,-4.92, P<0.05). Results of immunohistochemical staining: the cytomembrane and /or cytoplasm stained yellow, brown or sepia were defined as positive performance of CXCR3, CCR5 and CCR3. The absorbance A values of CXCR3 and CCR5 were(81.7±24.4)×10 -3 and (3.6±1.3)×10 -3 in the hypersplenic group,  which were significantly different from (19.2±5.8)×10 -3 and (1.2±0.4)×10 -3 in the control group (t=16.22, 9.09, P<0.05). The absorbance A value of CCR3 was (8.8±3.7)×10 -3 in the hypersplenic group and (7.9± 2.8)×10 -3 in the control group, respectively, showing no significant difference between the 2 groups (t=0.87, P>0.05). The rates of positive cells of CXCR3 and CCR5 was 52%±9% and 19%±5% in the hypersplenic group, which were significantly different from 21%±5% and 10%±3% in the control group (t=17.31, 8.21, P<0.05). The rates of positive cells of CCR3 in the hypersplenic group and control group were 35%±9% and 33%±14%, respectively, showing no significant difference between the 2 groups (t=0.43, P>0.05). Results of Western blot test: the relative expressions of CXCR3 and CCR5 were 2.45±0.85 and 0.94±0.48 in the hypersplenic group, which were significantly different from 1.31±0.95 and 0.32±0.26 in the control group (t=2.62, 2.91, P<0.05). The relative expression of CCR3 was 0.47±0.27 in the hypersplenic group, which was significantly different from 0.92±0.67 in the control group (t=-2.18, P<0.05).
Conclusion:The abnormal  expression of chemokine receptors in the spleen tissues of rats with cirrhosis and hypersplenism induced by CCl4 suggests that functional imbalance of Th1/Th2 lymphocyte subsets may play an important role in the regulation of peripheral blood cytopenia.