Roles And Mechanisms Of TNFAIP8L2 In Liver Fibrosis

ObjectiveIn recent years, more and more countries faced with liver fibrosis trouble. Liver fibrosis has become an important global health problem. To study and reveal the exact molecular mechanism has become an important topic of the liver fibrosis. The formation of liver fibrosis is a dynamic process. All kinds of damages result in the inflammation of the body, then the inflammation activates the cells to secrete chemokines and TGF-β1 to induce the hepatic stellate cells into fibroblast. Eventually, fibroblast proliferation makes the extracellular matrix such as collagen and proteogly excessive deposition and results in liver fibrosis. As we all know, Smad signaling pathway is thought to be the classical pathway for liver fibrosis caused by TGF-β1. We can inhibit liver fibrosis effectively by inhibiting Smad signaling pathway. Research and reveal the molecular mechanism of liver fibrosis is very important.The tumor necrosis factor-alpha-induced protein 8 (TNFAIP8) family is consist of TIPE (TNFAIP8), TIPE1 (TNFAIP8L1, TNF-α-induced protein 8-like-1), TIPE2 (Tumor necrosis factor-a-induced protein 8-like-2, TNFAIP8L2) and TIPE3 (TNFAIP8L3). They have homologous sequences. TIPE2 can inhibit the inflammatory response and maintain the immune homeostasis as a negative regulator of innate immunity and cellular immunity.A variety of reasons can cause inflammation which is the pathological basis of liver fibrosis. Therefore, the inflammation of liver tissues often plays a crucial role in the liver fibrosis. However, whether TIPE2 plays a role in the process of liver fibrosis has not been reported. Therefore, TIPE2-/- mice and C57 mice were used to establish hepatic fibrosis animal models and TGF-β1 was used to induce LX-2 cells to study the roles and mechanisms of TIPE2 during liver fibrosis.Methods1. To study the roles of TIPE2 in CCl4-induced liver fibrosis models in vivo1.1 To establish CCl4 induced liver fibrosis modelsMale C57BL/6 mice about six to eight weeks were randomly divided into two groups:the control group (n=20) were given Olive Oil about 5μl/g intraperitoneal injection twice a week. While the fibrosis model group (n=20) were given 20% CCI4 about 5μl/g intraperitoneal injection twice a week. In the 4th week, blood samples were collected by removing eyeball and centrifuged at 4000 rpm for 10 min to separate the serum for measurement of cytokines. The levels of murine MCP-1, TNF-a and IL-6 were quantified in the sera by Flow cytometry, and TGF-β1 was detected by ELISA. The levels of AST and ALT in the plasma were measured on a SpectraMax Plus spectrophotometer. In the 2th and 4th week, after the mice to death, one part of fresh liver tissues were conserved at -80℃ to perform molecular biological detection. A preparation of liver tissues were collected (perfusion by PBS) and were fixed with 4% buffered formalin, embedded in paraffin and then stained with hematoxylin and eosin (HE) and Masson for collagen deposition. Comprehensive judge were performed for the formation of liver fibrosis.1.2 To detect the expression of TIPE2 in liver fibrosis modelsEndogenous peroxidase activity of the liver tissues was quenched by incubation in 0.3% H2O2. And the liver tissues were blocked with goat serum. Rabbit anti-TIPE2 (JinSiTe,1:200), rabbit anti-α-actin (Abcam,1:200), rabbit anti-TGF-β1 (Abcam, 5μg/ml) were used for the first Abs. SP-9000 (ZSGB-BIO) was used for the secondary Abs for immunohistochemical analysis. The fresh liver tissues were used for western blot analysis to detect the expression of TIPE2 protein. The relationship between the degree of liver fibrosis and the levels of TIPE2 protein was analyzed.1.3 To verify the roles of TIPE2 in liver fibrosis by inducing TIPE2-/- models.Male C57BL/6 about six to eight weeks (n=10) and TIPE2-/- mice (n=10) were given 5% CCl4at 10 μl/g intraperitoneal injection once a week. In the 4th week, blood samples were collected by removing eyeball and centrifuged at 4000 rpm for 10 min to detect the levels of TNF-a, MCP-1 and 1L-6 and other cytokines in the serum. Liver samples were collected (perfusion by PBS) for Masson staining to analysis the liver fibrosis.1.4 Survival analysisMale C57BL/6 (six to eight weeks) (n=10) and TIPE2-/- mice (n=10) were given 20% CCl4 about 20μl/g intraperitoneal injection once a week. The mice living conditions were observed every day, and the changes of body weight and death of the mice were recorded. In the 12th week, mice were executed by conventional methods, and the survival curve was drawn.2. Studies In vitro2.1 Cell culture of LX-2The hepatic stellate cell line LX2 (GuangZhou Jennio Biotech Co., Ltd) were cultured in RIPA1640 supplemented with 10% FBS (GIBCO, Invitrogen), under the condition of a humidified air containing 5% CO2 at 37℃.2.2 To detect the expression of TIPE2 in LX-2LX2 cells were seeded in a six-well plate (4×105 cells/well) and incubated for 14 h before the addition of the stimulus. Different concentrations of TGF-β1 (0,2.5,5, 10,20ng/ml) were added to stimulate a certain period of time, and then using the methods of western blotting and real-time PCR to detect the expression of TIPE2 in protein level and mRNA level to determine the best concentration of TGF-β1. Then, using the best concentration (10ng/ml) of TGF-β1, the cells were stimulated for 0,12, 24 and 48 hours respectively to detect TIPE2 expression and determine the best work time of TGF-β12.3 To study the mechanisms of TIPE2 on liver fibrosis in LX-2 cellsLX2 cells were seeded in a 60 mm small dish (1×106 cells/dish) and incubated for 24 h, then transfected with TIPE2 expression plasmid pRK5-hTIPE2. The expression of a-actin and MMP-2 were detected by the methods of western blotting and real-time PCR to confirm the roles of TIPE2 in liver fibrosis. To study the mechanism of TIPE2 on liver fibrosis, the phosphorylation levels of Smad3、ERK、 IκB and JNK were detected by western blotting in TIPE2 transfected LX-2 cells stimulated with TGF-β1.2.4 To study whether TIPE2 regulates Smad signaling pathway in a Racl dependent-manner.Studies suggest that TIPE2 is an inhibitor of Racl. To study whether TIPE2 regulates Smad pathway in a Rac1 dependent-manner, we utilize Rac1 inhibitor (NSC23766) to study the activation of Smad3.Results1. The results in mice models1.1 TIPE2 is up-regulated markedly in the hepatic fibrosis modelsWe examined the hepatic fibrosis by HE and Masson staining. There is a large number of infiltrating inflammatory cells and the structure of hepatic lobule was damaged in the hepatic fibrosis model. Moreover, the collagen deposition in liver tissues was significantly increased in the hepatic fibrosis models compared with the liver tissue of normal control mice. The levels of hepatic fibrosis indicator a-actin and inflammatory factors (MCP-1, TNF-α, IL-6) were increased significantly in the hepatic fibrosis models, The MMP-2 was decreased significantly in the hepatic fibrosis models. These results suggested that the liver fibrosis models were successfully established. The expression of TIPE2 was detected using immunohistochemistry and western blotting in the liver tissues of hepatic fibrosis models. The results showed that TIPE2 protein was increased significantly in the hepatic fibrosis models than that in controls. Importantly, there was a positive correlation between the expression of TIPE2 and the degree of liver fibrosis. These results indicate that TIPE2 plays an important role during liver fibrosis.1.2 TIPE2-deficieny accelerates liver fibrosisWe performed a comparative analysis on liver fibrosis degree by Masson staining between C57 and TIPE2-/- mice models. The results showed that the inflammation of the liver tissues was obviously serious in the TIPE2-/- mice than that in C57 controls. Furthermore, the collagen deposition was significantly stronger in the TIPE2-/- mice than that in C57 mice. The serum cytokine levels (TNF-α and MCP-1) was also significantly higher in the TIPE2-/- mice than that in C57 controls. These results suggest that TIPE2 may play a protective role in liver fibrosis.1.3 The survival rate of C57 mice was higher than that in TIPE2-/- miceC57 mice and TIPE2-/- mice were given equivalent amounts of CCl4 twice a week. We comparative analysis the survival rates of C57 and TIPE2-/- mice. The results showed that C57 mice could live longer than TIPE2-/- mice.2. The results in cell line2.1 TIPE2 was up-regulated markedly in LX-2 cells stimulated with TGF-β1Western blotting and real-time PCR methods were used to detect the expression of TIPE2 in LX-2 cells stimulated with TGF-β1. Results showed that TIPE2 was upregulated markedly in TGF-β1 stimulated LX-2 cells. Moreover, the expression of TIPE2 was in a time-dependent and concentration-dependent manner.2.2 The expression of α-actin was down-regulated while MMP-2 was up-regulated in TIPE2 overexpressed LX-2 cells stimulated with TGF-β1.We used the western blotting and real-time PCR method to detect the expression of TIPE2 in LX-2 cells at protein level and mRNA level. The results showed that TIPE2 could down-regulated α-actin and upregulated MMP-2 in TGF-β1-stimulated LX-2 cells.2.3 TIPE2 could suppress Smad3、IκB and JNK signaling pathways in LX-2 cells stimulated with TGF-β1Smad signaling pathway and the NF-κB signaling pathway play important roles in liver fibrosis. To explore the mechanisms of TIPE2 on liver fibrosis, the levels of p-Smad3、p-ERK、p-IκB and p-JNK were detected in TIPE2 overexpressed LX-2 cells. The results showed that TIPE2 could down-regulate the levels of p-Smad3、 P-IκB and p-JNK markedly in TGF-β1stimulated LX-2 cells.2.4 TIPE2 suppresses Smad signaling pathway in a Racl-dependent mannerTIPE2 can inhibit the NF-κB and MAPK signaling pathways by combining with the Rac. To explore whether TIPE2 suppresses Smad signaling pathway in a Racl-dependent manner during liver fibrosis, Racl inhibitor was added in the LX-2 cells transfected with TIPE2 plasmids to detect Smad3 levels using western blotting method. The results showed that the inhibition effect of TIPE2 on the phosphorylation of Smad3 could be blocked by Rac1 inhibitor.Conclusions1.TIPE2 plays a protective role in the liver fibrosis formation.2.TIPE2 inhibits the formation of liver fibrosis by negatively regulating Smad signaling pathway in a Racl-depentent manner.Innovations and significances1.It is the first time to indicate that the anti-inflammatory protein TIPE2 plays a protective role in mice liver fibrosis models.2.TIPE2 inhibits the formation of liver fibrosis by negatively regulating Smad signaling pathway in a Racl-depentent manner.3. The results of this study could provide a new regulatory molecule for the liver fibrosis. Also, it can provide a new strategy for clinical treatments of liver fibrosis.