Activated hepatic stellate cells (HSCs) release pro-inflammatory and pro-fibrogenic factors. revealed that CXCL1 expressed in both hepatocytes and non-parenchymal liver cells (Physique 1B,C). Furthermore, an increase of CXCL1 cytoplasm expression was observed in the activated HSCs that were positive for -SMA in CCl4-induced mouse liver tissues (Physique 1C). Open in a separate window Physique 1 CXCL1 expression increased in liver fibrosis. (A) Real-time RT-PCR detection of hepatic gene expression of in normal control and carbon Rabbit polyclonal to Ki67 tetrachloride (CCl4)-treated mice (= 5). was used as the normalization control, ** 0.01; (B) Immunohistochemistry ABT-888 inhibitor analysis of CXCL1 in liver tissues from CCl4-induced mice; (C) Immunofluorescence detection of CXCL1 and -SMA in liver tissues from normal control and CCl4-induced mice (eight weeks). Arrows show CXCL1 (green) expression in the activated hepatic stellate cells (HSCs), which are positive for -SMA (reddish). 2.2. CXCL1 Promoted HSCs Activation and Co-Localized with CD147 in HSCs To evaluate the effect of CXCL1 on HSCs activation, LX-2 cells were treated with human recombinant CXCL1 (rCXCL1) for 24 h and subjected to detection of -SMA and type I collagen expression. Fluorescence activated cell sorting (FACS) and RT-PCR analysis showed that this expressions of -SMA and were increased with rCXCL1 activation (Physique 2A,B). Cell contraction assay exhibited that the surface area of gel was decreased (Physique 2C), indicating the cell rigorous contraction after rCXCL1 treatment. The proliferation of LX-2 cells was also promoted as measured with CCK-8 assay (Physique 2D). In the mean time, the activated HSCs showed both higher CD147 and CXCL1 expression (Physique 2E). Taken together, these results show that rCXCL1 promotes the activation phenotypes of HSCs. Open in a separate window Physique 2 CXCL1 promoted HSCs activation and co-localized with CD147 in ABT-888 inhibitor HSCs. (A) Circulation cytometry analyses of -SMA expression; (B) Real-time RT-PCR detection of mRNA level. was used as the normalization control; (C) Representative phase contrast images and quantitative analysis of collagen-based cell contraction; (D) CCK-8 assay; (E) Immunofluorescence detection of CD147, CXCL1 and -SMA in liver tissues from normal control and CCl4-induced mice (eight weeks, = 5). Arrows show -SMA (blue), CD147 (green), and CXCL1 (reddish) expression in the activated HSCs. LX-2 cells were treated with 100 ng/mL rCXCL1 for 24 h. The results were shown as the mean SD. * 0.05, ** 0.01, *** 0.001. 2.3. Generation of HSCs-Specific CD147-Knockout Mice We hypothesize that CD147 regulates the CXCL1 expression in HSCs. To obtain HSCs-specific CD147-knockout mice, we crossed the conditional CD147 targeting mice (transgenic mice. Four types of transgenic mice and were generated (Physique 3A). The and mice were used for the following experiments. Histological analysis revealed that mice showed no spontaneous lesions in lung, heart, kidney, spleen, testis, liver and brain (Physique 3B). It was reported that GFAP mainly expresses on astrocytes in the central nervous system, while also expressing in the cartilage cells, fibroblast, hepatic epithelial cells and HSCs [14,15,16]. The mice showed the lower expression of CD147 in brain and liver both in the mRNA and protein levels, while there was no such significant switch in other tissues (Physique 3C,D). The primary HSCs were then isolated to further verify the specific knockout of CD147 in mouse HSCs. Western blot and RT-PCR analysis showed that this expression of CD147 in isolated HSCs from mice was significantly reduced compared with that of mice (Physique 3E). ABT-888 inhibitor Open in a separate window Physique 3 Generation of HSCs-specific CD147-knockout mice. (A) Identification of specific knockout of gene in the mouse genome; (B) HE stain of different tissues in and was used as the normalization control. The results were shown as the mean SD. = 3. * 0.05, ** 0.01, *** 0.001. 2.4. CD147 Deletion in HSCs Alleviated CCl4-Induced Liver Fibrosis and Deregulated CXCL1 Expression The and mice were subjected to CCl4 intraperitoneal injection for induction of liver fibrosis. According to the anatomical structure, the mouse liver was divided into the papillary lobe, caudate lobe, right lobe, ABT-888 inhibitor left lobe (up), left lobe (down), right middle lobe, and left middle lobe (Physique 4A). The histological images showed that mice experienced obvious pseudolobule and infiltration of inflammatory cells, whereas mice showed attenuated pseudolobule coupled with the reduced infiltration of inflammatory cells and liver damage (Physique 4B). The collagen was stained with sirius reddish, and the expression intensity and the percentage of positive expression area were statistically analyzed. As shown in Physique 4C, mice.