Effects Of Locostain On Collagen Metabolism Of Liver Fibrosis

Hepatic fibrosis (HF) is the common pathological changes of chronic liver injury which can be induced by many pathogenic factors, such as virus, alcohol, chemical drugs and so on. Those sustained factors lead to the excessive deposition of the extracellular matrix (ECM) and abnormal production of fibrillar connective tissue. HF is an inevitable stage from chronic hepatitis to cirrhosis and liver cancer. Therefore, prevention, treatment and even reversal of liver fibrosis have become the focus worldwide.Hepatic stellate cells (HSCs) are associated with HF. HSCs would be activated if injury and stimulation affect on liver. The phenotype of HSCs will transform from quiescence to activation, which is called myofibroblast cells (MFCs). The abnormal deposition of ECM caused by MFCs leads to the formation of hepatic fibrosis and reconstruction of inner liver structure. The main components of ECM are collagens. Type Ⅰ and Ⅲ collagen increase significantly in hepatic fibrosis. Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) regulate collagen metabolism. Therefore, the activation of HSCs is the centre step of liver fibrosis.Hepatic fibrogenesis is accompanied by the activation of Raf-1 /MEK/ERK1,2 pathway, activation of this signaling pathway promotes HSCs proliferation, differentiation and migration. Raf kinase inhibitor protein (RKIP) is a member of phosphatidylethanolamine binding proteins (PEBPs) family. RKIP present in plants and animals, and can regulate many signalings trans-duction. RKIP interacts with Raf-1 kinase domain, suppressing the transduction of the Raf-1/MEK/ERK1,2. The overexpression of RKIP can promote the migration of HSCs. Locostation, an ephedroxane derivative without antimicrobial effects, is a specific inhibitor of RKIP. Locostation can promote HSCs activation and suppress its migration by restraining the expression of HSCs.Objective:Aming to study the effect of Locostatin on liver fibrosis, the mice underwent hepatic fibrogenesis induced by CCl4 were treated with intraperitoneal injected Locostatin.Methods:Mice were grouped as follows:(1) Normal group; (2) CC14 induced model group; (3)Locostatin treatment group. During the process of modeling, the mice of treatment group accept intraperitoneal injection of Locostatin. The content of alanine aminotransferase (ALT), aspartate transaminase (AST) and hydroxyproline (HYP) in serum were detected. Pathological changes in the liver were measured by HE staining. Masson and sirus-red staining of hepatic tissue were used to measure the changes of collagens. RT-qPCR was used to detect the mRNA expression of type Ⅰ, Ⅲ and Ⅳ collagen, MMP-13, MMP-2, TIMP-1, TIMP-2, RKIP and α-SMA. Immunohistochemistry (IHC) and western blot were used to determine the content of type Ⅰ, Ⅲ and Ⅳ collagen, α-SMA and RKIP. MMP-13, MMP-2, TIMP-1, TIMP-2 expressions were detected by enzyme-linked immune-sorbent assay (ELISA).Results:1 Pathological staining showed that Locostatin could inhibit inflammation, the activation of HSCs and the deposition of collagen. In HE staining, the inflammation of model group more serious than treatment group, and the normal group had no inflammation. In the Masson and sirus-red staining, the collagen deposition in model group increased compared with the treatment group. The normal group had no abnormal collagen deposition. In the result of IHC, the content of type Ⅰ collagen, type Ⅲ collagen and α-SMA in model group increased than the treatment group. In the normal group the expressions of type Ⅰ collagen, type Ⅲ collagen and α-SMA were the least. The content of type Ⅳ collagen in normal group increased compared with the model group, but decreased compared with the treatment group. The content of ALT in model group (189.43±64.25) increased compared with normal group (8.06±4.46) and treatment group (61.88±5.40) (P<0.01). Normal group and treatment group had no difference (P>0.05). The content of AST in model group (231.15±39.86) increased compared with normal group (39.46±23.11) and treatment group (84.61±29.36) (P<0.01). There was no significant difference (P>0.05) between normal group and treatment group. The content of HYP in model group (3.07±0.47) increased compared with normal group (1.63±0.78) and treatment group (2.06±0.83) (P<0.01). Normal group and treatment group had no difference (P>0.05).2 Locostatin could decrease the mRNA expressions of α-SMA, type Ⅰ and type Ⅲ collagen, increase the mRNA expressions of RKIP and type Ⅳ collagen.2.1 Locostatin could decrease the mRNA expression of type Ⅰ and type Ⅲ collagen. The mRNA expression of type Ⅰ collagen and type Ⅲ collagen in model group (9.51±1.37,3.23±0.43) increased compared with the treatment group (3.60±0.31,1.25±0.22) (P<0.01).2.2 Locostatin could increase the mRNA expression of type Ⅳ collagen. The mRNA expression of type Ⅳ collagen in model group (0.71±0.21) decreased compared with the treatment group (4.80±1.22) (P<0.01).2.3 The mRNA expressions of TIMP-1, MMP-13, TIMP-2 and MMP-2 in model group(0.84±0.14,0.91±0.02,0.45±0.05,0.73±0.25) had no difference with the treatment group(0.79±0.13,0.60±0.22,0.49±0.18,0.58±0.16) (P>0.05).2.4 Locostatin could decrease the mRNA expression of α-SMA. The mRNA expression of α-SMA in model group (6.73±0.10) increased compared with the treatment group (2.71±0.31) (P<0.01).2.5 Locostatin could increase the mRNA expression of RKIP. The mRNA expression of RKIP in model group (1.06±0.16) decreased compared with the treatment group (3.49±0.50) (P<0.01).3 Locostatin could decrease the protein expressions of α-SMA, type Ⅰ and type Ⅲ collagen, increase the protein expressions of RKIP and type Ⅳ collagen.3.1 Locostatin could reduce the deposition of type Ⅰ collagen. The expression of type Ⅰ collagen in treatment group (0.68±0.06) increased compared with the normal group (0.22±0.04) (P<0.01). The expression in treatment group decreased compared with the model group (1.37±0.13) (P<0.01).3.2 Locostatin could reduce the deposition the of type ⒊ collagen. The expression of type Ⅲ collagen in treatment group (0.16±0.02) increased compared with the normal group (0.11±0.02) (P<0.01). The expression in treatment group decreased compared with the model group (0.26±0.07) (P<0.01).3.3 Locostatin could increase the expression of type Ⅳ collagen. The expression of type Ⅳ collagen in treatment group (0.21±0.04) decreased compared with the normal group (0.38±0.07) and treatment group (0.68±0.04) (P<0.01).3.4 CC14 could increase the expression of α-SMA and active HSCs. The expression of α-SMA in model group (1.54±0.13) increased compared with the normal group (0.42±0.04) and treatment group (0.63± 0.06) (P<0.01). The expression of α-SMA in treatment group increased compared with the normal group (P<0.05).3.5 CCl4 couldn’t affect the expression of RKIP, but Locostatin could increase the content of RKIP. The expression of RKIP in treatment group (0.82±0.12) increased compared with the model group (0.46±0.09) (P<0.01). The normal group (0.29±0.05) and model group had no difference (P>0.05).4 Locostatin could regulate the ratio of MMP-13 to TIMP-1, and decrease the degradation of type Ⅰ collagen and type Ⅲ collagen.4.1 CCl4 could decrease the expression of TIMP-1, but Locostatin had no effect on the expression of TIMP-1. The expression of TIMP-1 in model group (136.88±39.51) and treatment group (140.39±24.70) decreased compared with the normal group (244.29±74.25) (P<0.05). The model group and treatment group had no difference (P>0.05).4.2 Locostatin had no effect on the expression of MMP-13, but CC14 could decrease the expression of MMP-13. The expression of MMP-13 in model group (23.04±4.05) and treatment group (20.44±5.35) decreased compared with the normal group (31.96±8.28) (P<0.05). The model group and treatment group had no difference (P>0.05).4.3 CCl4 could decrease the expression of TIMP-2 whereas Locostatin had no effect on the expression of TIMP-2. The expression of TIMP-2 in model group (8.72±5.17) and treatment group (9.42±3.01) decreased compared with the normal group (18.07±7.18) (P<0.05). The model group and treatment group had no difference (P>0.05).4.4 CCl4 could decrease the expression of MMP-2 but Locostatin had no effect on the expression of MMP-2. The expression of MMP-2 in model group (326.42±64.63) and treatment group (342.54±66.73) decreased compared with the normal group (700.99±245.48) (P<0.05). The model group and treatment group had no difference (P>0.05).4.5 Locostatin could increase the ratio of MMPs to TIMPs, and promote collagen degradation. The ratio of MMP-13 to TIMP-1 in model group (0.17±0.02) and treatment group (0.19±0.01) increased compared with the normal group (0.13±0.02) (P<0.01), the model group decreased compared with the treatment group (P<0.01).4.6 The ratio of MMP-2 to TIMP-2 in model group (0.39±0.02), treatment group (0.43±0.03) and normal group (0.40±0.07) had no difference with each other (P>0.05)Conclusion:Locostatin could reduce mice liver inflammation, this maybe because of Locostatin inhibite the migration of inflammatory cells. Secondly, by inhibiting the activation of HSCs and increasing the the ratio of MMP-13 to TIMP-1, Locostatin could decrease the deposition of type Ⅰ and type Ⅲ collagen, and delay the formation of mice liver fibrosis.