| Objectives:To investigate expression and significance the of CXCL12-CXCR4/CXCR7 chemokine axis in the spleens of rats with cirrhosis and hypersplenism, provide theoretical basis for the further explore the mechanism of spleen fibrosis of cirrhosis and hypersplenism.Methods:Fifty male inbred SD rats were randomly divided into a model group (n= 40) and a control group (n= 10). The rats in the model group were first gavaged with 40% CCL4/peanut oil solution(0.3ml/100g,twice per week) and 15%white spirit for 8 weeks to develop cirrhosis and hypersplenism.The rats in the control group received normal saline(0.3ml/100g (twice per week for 8 weeks). The animal models with cirrhosis and hypersplenism were confirmed by liver function test, routine test, HE staining and Masson’s trichrome staining after visual inspection. The short-axis long-axis diameter of spleen and spleenindex were also examined.The fibrosis level of spleen tissue was observed by Masson’s trichrome staining. The expression of the CXCL12-CXCR4/CXCR7 chemokine axis in the spleen was measured by immunohistochemistry, Western blotting, and qRT-PCR. Measurement data with normal distribution were presented as x±s. Comparison between the two groups were used the independent sample T test.Results:Changes of liver function:the level of ALT,AST,TBIL and TP of rats were(237.80±105.11)U/L,(685.70±246.47)U/L,(30.04±8.77)μmol/L,(53.45±6.41)g/L in the model group, which were significantly different from(26.57±7.21) U/L,(123.43±18.64) U/L,(4.07±3.45)μmol/L,(64.97±2.72)g/L in the control group(t=6.333,7.185,7.383,-4.449,P<0.05). Changes of peripheral blood cell count: The RBC, PLT, Hb count of the models were(5.99±1.81)×1012/L,(412.00±260.46) ×109/L,(107.82±32.27) g/L, which were inferior (t=-2.856,-7.234,-2.996, P<0.05)to the control((8.04±0.61) ×1012/L,(1108.71±147.25) ×109/L,(138.00±6.90)g/L), but the WBC count in the models was superior (t=5.256, P<0.05)to the control ((24.68±11.34) ×109/L vs. (6.18±2.21) ×109/L). The spleen index and the two diameters of the spleen were significantly enlarged in models compared with the controls((3.14±0.98) mg/g vs. (2.33±1.03) mg/g, (0.92±0.08) cm vs. (0.70±0.09) cm, (4.24±0.27) cm vs. (3.69±0.24) cm,t=2.351,6.784,5.399,P<0.05).Masson trichrome staining displayed that collagen fiber area in the spleen of rats induced by CCL4 is remarkably larger than the controls((11.17±4.85)% vs. (2.83±0.90)%, t=7.939,P<0.01).Immunohistochemical staining revealed that the mean density of CXCL12, CXCR4 and CXCR7 was significantly increased in models compared with the controls((8.89±4.89)×10-3vs.(2.89±1.11)×10-3,t=4.126,P<0.011;(8.78±4.67)×10-3vs.(2.03 ±0.45)×10-3,t=4.973,P<0.01;(8.47±3.97)×10-3vs.(1.81±0.69)x10-3,t=5.644,P<0.01;respec tively). Meanwhile, the models displayed a higher rate of positive cells of CXCL12,CXCR4 and CXCR7 ((31.02±9.03)%vs. (20.62±8.88)%, t=3.112,P<0.01;(33.22±8.31)%vs. (18.81±6.23)%, t=4.170,P<0.01; (28.89±8.40)% vs. (10.49±2.99)%, t=6.293, P<0.01;respectively). Moreover, western blot analysis showed that the protein expression of CXCL12, CXCR4 and CXCR7 in the models were higher than that in the controls (1.71±0.98 vs.0.39±0.13, t=6.366,P<0.01; 1.01±0.57vs. 0.32±0.11, t=5.550,P<0.05; 1.15±0.77 vs.0.51±0.31, t=3.348,P<0.01;respectively).In addition, qRT-PCR demonstrated that, as compared with the controls the mRNA expression of CXCL12,CXCR4 and CXCR7 were improved prominently in the models(3.48±0.90vs.1.05±0.10,t=9.607,P<0.01; 1.99±0.34 vs.1.00±0.18, t=10.460,P<0.01; 2.53±0.59vs.1.02±0.02, t=9.259, P<0.01; respectively). Besides, positive correlations were found between the protein expression of CXCL12,CXCR4 and CXCR7 and splenic collagen fiber area(r=0.688, P<0.01;r=0.711, P<0.01;r=0.579, P<0.01; respectively),and then the mRNA expression of CXCL12,CXCR4 and CXCR7 were also closely correlated with the splenic collagen fiber area(r=0.694, P<0.01;r=0.647, P<0.01;r=0.609, P<0.01; respectively).Conclusions:The abnormally increased expression of CXCL12-CXCR4/CXCR7 in the enlarged spleen of rats with cirrhosis and hypersplenism caused by CCL4 indicated severe fibrosis in the enlarged spleen and was positively correlated with the spleen fibrosis area. CXCL12-CXCR4/CXCR7 might be involved in the entire process of spleen cirrhosis in rats with cirrhosis and hypersplenism and potentially lead to peripheral cytopenias. |
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