Study Of Carbon Tetrachloride-induced Hepatic Injury On Zinc Finger Protein Zbtb20 Hepatocyte-specific Knockout Mice

Zbtb20 is a member of zinc finger protein family containing BTB/POZ and C2H2 domains. It has been suggested that Zbtb20 plays a variety of important roles in multiple systems. Zbtb20 mRNA was developmentally activated in postpartum liver reaching a plateau at the age of 8 weeks. To determine its physiological functions in vivo, we have previously generated hepatocyte-specific Zbtb20 knockout mice (LZB20KO) by the Cre/loxP approach and demonstrated that Zbtb20 functions as a transcriptional repressor regulating the developmental expression of the alpha-fetoprotein (AFP) gene in the liver. However, little is known about whether Zbtb20 plays any roles in additional aspects of liver functions such as drug metabolism and detoxification.To define whether Zbtb20 plays a role in liver-specific metabolic functions, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl4) in LZB20KO mice. We then examined the hnRNA, mRNA and protein expression level of cytochrome P4502E1 (Cyp2El), the major enzyme whose products underlie CCl4-induced injury. We also analyzed the mRNA expression level of hepatocyte nuclear factor la (Hnfl a) and the activation ofβ-catenin, which has been previously reported to regulate Cyp2e1 transcription. At last, we detected the serum insulin and L-3,3,5-triiodothyronine (T3) level, which has been shown to affect cyp2el mRNA stability. Our main findings are described as follows:1. LZB20KO mice were resistant to CCl4-induced acute hepatic damage.(1) Histological analysis data showed that the peak of injury was reached 24 h after a single dose of CCl4 injection in both LZB20KO and control mice. However, the LZB20KO mice exhibited attenuated liver injury by showing a significant decrease in liver necrosis area and accelerated hepatolobular repair. (2) LZB20KO livers showed a significant decrease in TUNEL+apoptotic cells 72h after CCl4 administration when compared with control mice. (3) Serum ALT and AST levels in LZB20KO mice were also significant lower than that in control mice 24,48 and 72h after CCI4 challenge, which paralleled the histopathological findings. There were no significant differences in histology, apoptotic cells and serum ALT and AST levels between LZB20KO and control mice after olive oil (serve as vehicle) injection. These data indicate that LZB20KO mice were less sensitive to CCl4-induced acute hepatic damage.2. Accelerated hepatolobular repair after CC14 administration in LZB20KO mice was not due to an increased proliferation.Hepatocyte proliferation was determined by BrdU incorporation assay. A relatively small number of BrdU+cells were scattered in the liver in both LZB20KO and control mice 24h after CCl4 challenge, with no significant differences between the two groups. The peak hepatocyte proliferation occurred at 48 h in both LZB20KO and control mice, but the number of BrdU+cells in LZB20KO mice was great lower than that in control mice, probably due to alleviated hepatic damage. This result was confirmed by immunohistochemical staining of Ki67 and PCNA, two proliferation markers. There were only a few BrdU+cells in both LZB20KO and control liver after olive oil injection, with no significant differences between the two, suggesting most cells are normally in the resting phase. Thus, the accelerated tissue repair after CCl4 exposure was not likely due to an increased proliferation in LZB20KO mice.3. LZB20KO mice exhibited an alleviated CCl4-induced chronic hepatic fibrosis.Fibrosis was induced by injection of CCl4 twice each week for 6 weeks. Livers were then subjected to picro-sirius staining for showing collagen I deposition. The staining showed that collagen accumulation was greatly subdued in LZB20KO mice as compared with that in control mice. Thus, LZB20KO mice were also resistant to CCU-induced chronic hepatic fibrogenesis.4. The expression of Cyp2El mRNA and protein was down-regulated in LZB20KO liver.In order to reveal the mechanism underlying LZB20KO mice'resistance to CCl4 hepatotoxicity, we analyzed the expression of Cyp2E1, a key enzyme responsible for the activation of CCl4 in livers. The relative expression level of Cyp2El protein in LZB20KO liver was decreased by 60%determined by Western Blot, as compared to those in controls. Immunohistochemistry showed that CYP2E1 were still expressed in centrilobular pattern, but the positive area was signifivantly decreased in LZB20KO liver compared to control livers. The relative expression level of Cyp2El mRNA in LZB20KO liver was also decreased by 50%, while the expression of Cyp2E1 hnRNA was not changed, as determined by Real-time quantitative PCR. This data indicate that the down-regulation of Cyp2E1 mRNA in LZB20KO livers occurs most likely at post transcriptional levels.5. Normal expression of Hnfla and activation ofβ-catenin in LZB20KO liver.To reveal the mechanism underlying the down-regulation of Cyp2El mRNA expression in LZB20KO liver, we examined the mRNA expression of Hnfla.which is a key activator for Cyp2El transcription. There was no significant difference in Hnfla mRNA levels between LZB20KO and control livers. We next analyzed the activation ofβ-catenin, which has been previously reported to regulate Cyp2el transcription. No obvious difference was observed between LZB20KO and control livers. These data suggest that the down-regulation of Cyp2E1 mRNA expression in LZB20KO liver occurs most likely at post transcriptional levels.6. Normal serum insulin level and expression of spot14 mRNA in LZB20KO liver.To investigate possible post-transcriptional mechanism underlying the down-regulation of Cyp2E1 mRNA expression in LZB20KO liver, we detected the serum insulin and T3 level, which has been shown to affect cyp2el mRNA stability. There was no significant difference in serum insulin level between LZB20KO and control livers. The serum T3 level was slightly higher but the expression of spot 14 mRNA, a T3 responsive gene, was not changed in LZB20KO when compared to control mice.In conclusion, we showed that the liver-specific ablation of Zbtb20 resulted in the down-regulation of Cyp2El mRNA and protein expression. Consequently, these mice were resistant to CCl4-induced acute hepatic damage and chronic fibrosis. The down-regulation of Cyp2E1 mRNA in LZB20KO livers occurs most likely at post transcriptional levels.These findings strongly suggest that in addition to regulating AFP transcription, Zbtb20 may also control drug metabolism and detoxification in the liver.