Hedgehog Signaling Pathway And Drug In A Mouse Model Of Liver Fibrosis Intervention Exploration

Section oneThe dynamic activation pattern of hedgehog signaling pathway in carbon tetrachloride induced liver fibrosis in miceBackground and aims:Liver fibrosis is a common pathological process of chronic liver injury caused by a variety of etiologies, characterized by excessive production, and deficiency in degradation of extracellular matrix, which leads to deposition of extracellular matrix in the liver, leading to liver cirrhosis. Besides the parenchyma cells within the liver, many immunological cells, as well as a plethora of cytokines are getting involved in the complicated process of liver fibrosis. Recent studies have suggested a possible role of hedgehog (Hh) signaling pathway in the development of liver fibrosis. We set to explore the dynamic activation pattern of hedgehog signaling pathway in liver fibrosis by using the murine model of liver fibrosis induced by carbon tetrachloride (CCl4).Methods:Thirty-two male C57BL/6J mice (7-8weeks old) were randomly assigned to three groups:the normal control group (n=12), the model group (n=12), and the spontaneous regression group (n=8). For the induction of liver fibrosis murine model, mice were injected with0.8ml/kg CCl4intraperitoneally, twice a week. The spontaneous regression group received CCl4for4weeks, as did the model group, but then stopped receiving intraperitoneal CCl4for the following2weeks. Serum and fresh liver specimens were collected periodically, at the end of week3and week6. HE staining and Masson staining techniques were used to evaluate the extent of liver fibrosis of diseased mice at different time points. Liver biochemical parameters and hydroxyproline (Hyp) content were also evaluated. The expression of a-smooth muscle actin (a-SMA), sonic hedgehog (Shh), and Glioblastoma(Gli) family transcription factors (Gli1, Gli2) were detected by immunochemistry, western blotting and real-time PCR.Results:Three weeks after CCl4injection, mice in the model group exhibited degeneration and necrosis of hepatocytes, disorganization of hepatic lobules, and deposition of excessive extracellular matrix were seen around the central veins and in the portal areas. These alterations exacerbated as mice received another three weeks’ CCl4injection. Mice in the spontaneous regression group had significantly less degeneration and necrosis of hepatocytes, and extracellular matrix around the central veins and in the portal areas were also markedly lessened. Mice receiving3and6 weeks’ injections of CC14both had increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), and Hyp compared to their healthy littermates (P<0.01). Of these, Mice receiving6weeks’ injections of CCl4had more severe liver biochemical parameters and Hyp content than those receiving3weeks’ injections of CCl4. Mice in the spontaneous regression group had comparable levels of ALT, AST, TBil, and Hyp with mice receiving3weeks’ injections of CC14, which were significantly lowered compared to mice receiving6weeks’ injections of CCl4. Immunochemical findings revealed that compared to their healthy littermates, mice receiving3or6weeks’injections of CCl4had markedly increased numbers of cells stained positive for Shh, Gli1, Gli2, and a-SMA. Moreover, the distribution of a-SMA(+) and Shh (+) cells seem to be consistent, both located mainly around the proliferated bile ducts, the fibrous septums and in the portal areas; while the distribution of Gli1(+) and Gli2(+) cells seem to be consistent, both located mainly around the interlobular veins and in the portal areas. However, Gli1(+) and Gli2(+) cells are seldom found around the fibrous septums. Mice in the spontaneous regression group had comparable numbers of cells stained positive for Shh, Gli1, Gli2, and a-SMA, with mice receiving3weeks’ injections of CCl4, which were significantly lessened compared to mice receiving6weeks’injections of CCl4. Western blotting and real-time PCR have consistently convinced that mice receiving3and6weeks’ injections of CCl4both had markedly increased expression of Shh, Gli1, Gli2, a-SMA, in terms of mRNA and protein levels, compared to normal mice. Mice in the spontaneous regression group had lowered expression of Shh, Gli1, Gli2, and a-SMA, in terms of mRNA and protein levels, compared to mice receiving6weeks’ injections of CCl4.Conclusion:We successfully reproduced liver fibrosis rodent model by injecting0.8ml/kg CCl4intraperitoneally into the male C57BL/6J mice (7-8weeks old), twice a week. Mice receiving6weeks’ injections of CCl4had consistently more severe liver fibrosis than those receiving3weeks’ injections of CCl4. Mice injected intraperitoneally with CCl4for4weeks, and then stopped receiving CCl4for the following2weeks experienced an auto-regression of live fibrosis, which were comparable or even milder than those in mice receiving3weeks’ injections of CCl4. Hedgehog signaling pathway were quiescent in the normal murine liver, but were activated in response to injection with CCl4. The expression of several Hh pathway molecules increased along with the progression of liver fibrosis, and then decreased along with the auto-regression of liver fibrosis. These data suggests a potentially important role of Hedgehog pathway activation in the development of liver fibrosis. Section twoThe preventative and therapeutic effects of cyclopamine and curcumin in CCl4induced liver fibrosis in miceBackground and aims:Cyclopamine is a specific inhibitor of Hedgehog (Hh) signaling pathway. Recent studies have suggested that the inhibition of pathological activation of Hedgehog signaling pathway may cause amelioration of liver fibrosis. Curcumin has long been recognized for its anti-fibrotic, anti-oxidative, and anti-inflammatory efficacy in a variety of chronic liver diseases. Curcumin has been evaluated in animal experiments, convincing its protective effects in early-stage liver fibrosis. However, for those with advanced liver fibrosis, the effect of curcumin is controversial. We set to determine whether cyclopamine and curcumin could ameliorate murine liver fibrosis induced by CCl4. Then, we intend to explore whether their protective effects are achieved by inhibiting the pathologically activated Hh signaling pathway.Methods:Seventy-two male C57BL/6J mice (7-8weeks old) were randomly assigned to8groups:the normal control group (n=12), the model group (n=12), low dose cyclopamine prevention group (n=8), high dose cyclopamine prevention group (n=8), curcumin prevention group (n=8), low dose cyclopamine treatment group (n=8), high dose cyclopamine treatment group (n=8),curcumin treatment group (n=8). For the induction of liver fibrosis murine model, mice were injected with0.8ml/kg CCl4intraperitoneally, twice a week. The low dose cyclopamine prevention group, high dose cyclopamine prevention group, and curcumin prevention group received5mg/kg/d cyclopamine,10mg/kg/d cyclopamine, and400mg/kg/d curcumin, respectively, for a duration of3weeks, concomitantly with the injection of CCl4. On the contrary, low dose cyclopamine treatment group, high dose cyclopamine treatment group, and curcumin treatment group received5mg/kg/d cyclopamine, lOmg/kg/d cyclopamine, and400mg/kg/d curcumin, respectively,3weeks later than the corresponding drug prevention groups, and for a duration of3weeks. Serum and fresh liver specimens were collected periodically, at the end of week3and week6. HE staining and Masson staining techniques were used to evaluate the extent of liver fibrosis in all the groups. Liver biochemical parameters and hydroxyproline (Hyp) content were also evaluated. The expression of a-smooth muscle actin (a-SMA), sonic hedgehog (Shh), and Glioblastoma(Gli) family transcription factors (Gli1, Gli2) were detected by immunochemistry, western blotting and real-time PCR.Results:Compared to mice receiving3weeks’ injections of CCl4without any drugs, mice in the low dose cyclopamine prevention group, high dose cyclopamine prevention group, and curcumin prevention group all had improved biochemical parameters, Hyp content, and histological findings. Immunochemical staining revealed that mice within all the drug prevention groups had markedly lessened numbers of cells stained positive for Shh, Gli1, Gli2, and a-SMA. Western blotting and real-time PCR have consistently convinced that mice within all the drug prevention groups had markedly decreased expression of Shh, Gli1, Gli2, a-SMA, in terms of mRNA and protein levels, compared to mice receiving3weeks’injections of CCl4without any drugs. Compared to mice receiving6weeks’ injections of CCl4without any drugs, mice in the low dose cyclopamine treatment group, high dose cyclopamine treatment group, and curcumin treament group showed similar biochemical parameters, Hyp content, and histological findings. Immunochemical staining revealed that mice within all the drug teatment groups had similar numbers of cells stained positive for Shh, Glil, Gli2, and a-SMA, compared to mice receiving6weeks’ injections of CCl4without any drugs. Western blotting and real-time PCR have consistently convinced that mice within all the drug treatment groups had similar expression of Shh, Gli1, Gli2, a-SMA, in terms of mRNA and protein levels, compared to mice receiving6weeks’ injections of CCl4without any drugs.Conclusion:Cyclopamine and curcumin may prevent and ameliorate liver fibrosis induced by CCl4in mice, which may be related to the inhibition of the pathologically activated Hh signaling pathway. The Hh pathway may be a new therapeutic target for treating liver fibrosis. Neither cyclopamine nor curcumin could cure advanced liver fibrosis, which suggested a careful selection of therapeutic time window should be considered in future studies.